Lateral Flow Analyte Detection

ABSTRACT

Lateral flow methods and apparatuses for detecting one or more analytes are provided. Certain embodiments provide a lateral flow device, kit and method of using the same, comprising: a flow path defined by a permeable sub-assembly of the lateral flow device, a release zone comprising a plurality of peptide-tagged agents, and a detection zone comprising a plurality of anti-peptide agents present in the flow path at a ratio of at least 100:1, on a weight:weight basis, relative to the plurality of peptide-tagged agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority from U.S. ProvisionalApplication No. 62/355,133, filed Jun. 27, 2016. The foregoing relatedapplication, in its entirety, is incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 21, 2016, isnamed 013018-0009-999_SL.txt and is 2,160 bytes in size.

FIELD OF THE INVENTION

The present disclosure relates to methods and apparatuses for detectingone or more analytes in a sample. In particular, this specificationprovides improved embodiments of lateral flow analyte detection methodsand apparatuses.

BACKGROUND OF THE INVENTION

Lateral flow assays combine various reagents and process steps in onecontainer, thus providing relatively convenient and rapid detection ofthe presence, or absence, of a variety of analytes in liquid samples,including antigens such as hormones, e.g., human chorionic gonadotrophin(hCG) for pregnancy testing, antibodies such as antibodies to infectiousagents, and haptens such as drugs of abuse.

Conventional lateral flow assays provide a permeable test stripconfigured to (a) expose an analyte-containing sample to a detectableagent which may bind with the analyte; and (b) passively transport(e.g., by capillary action) the analyte bound with the detectable agentto a detection zone of the test strip, where it is immobilized to thetest strip in a matter of minutes. The analyte bound with the detectableagent may be immobilized, for example, with an immobilization agentpresent on the test strip. To facilitate passive transport, the teststrip typically comprises one or more permeable materials in fluidcommunication.

A conventional lateral flow assay may be more expensive than acomparable plate assay employing the same type of immobilization agentbecause, inter alia, a sample may flow through the lateral flow teststrip under the influence of capillary action in a matter of minutes. Byway of comparison, a traditional plate assay may be configured toincubate the sample in the presence of an immobilization agent for amuch longer period of time typically between 30 minutes and 2 hours. Tocompensate for reduced contact time, lateral flow assays are oftenconfigured with a higher concentration of immobilization agent and/ordetectable agent, relative to comparable plate assays, in order toensure that sufficient immobilization agent may be present to capture atleast a detectable portion of the analyte, particularly when the analyteis expected to be present at low concentration in the sample. When theimmobilization agent is expensive, the cost difference can becomeconsiderable (for example, an assay quantity of a monoclonal antibodyimmobilization agent may cost significantly more).

The need for a high concentration of detectable agent in a lateral flowassay may also impact test sensitivity, particularly if excess unbounddetectable agent is difficult to wash from the test strip. Such excessunbound detectable agent may produce significant background noise thatinterferes with the detection signal.

In addition, fixing the immobilization agent and the other requiredassay components in a single combination on the test strip proscribesmixing and matching different assay component combinations, resulting inreduced flexibility and potential waste of assay components, for exampleif detection of a different type of analyte is required.

Accordingly, there is a need for lateral flow assays which are moreflexible, more sensitive, and/or less expensive.

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in any country.

BRIEF SUMMARY OF THE INVENTION

Certain embodiments may provide an assay platform for detecting ananalyte. In certain embodiments, the assay platform may comprise, forexample, a permeable zone that may be configured, for example, totransport at least a portion of a sample that may comprise the analyte,and optionally a further permeable zone may be configured, for example,to receive at least a portion of the sample. In certain embodiments, thepermeable zone may comprise, for example, a plurality of peptide-taggedagents. In certain embodiments, the plurality of peptide-tagged agentsmay be capable of forming an analyte complex that may comprise at leastone of the plurality of peptide-tagged agents and the analyte. Incertain embodiments, the further permeable zone may be configured toreceive the portion of the sample from the permeable zone. In certainembodiments, the further permeable zone may comprise, for example, aplurality of anti-peptide agents present, for example, ata ratio of atleast 100:1, for example at least 125:1, 200:1, or 300:1, on aweight:weight basis, relative to the plurality of peptide-tagged agents.In certain embodiments, the plurality of anti-peptide agents may beconfigured, for example, to immobilize the at least one of the pluralityof peptide-tagged agents. In certain embodiments, the plurality ofanti-peptide agents may be configured, for example, to immobilize theanalyte complex. In certain embodiments, the plurality of anti-peptideagents may be bound to at least a portion of the further permeable zone.In certain embodiments, at least one of the at least one of theplurality of peptide-tagged agents and at least one of the plurality ofanti-peptide agents may be binding partners.

In certain embodiments, the transporting may comprise, for example,passive transport, capillary action, wetting, wicking, or a combinationof two or more thereof.

In certain embodiments, the at least one of the plurality ofpeptide-tagged agents may be bound to at least a portion of the furtherpermeable zone. In certain embodiments, at least one of the at least oneof the plurality of peptide-tagged agents may comprise, for example, aplurality of covalently conjugated peptide tags. In certain furtherembodiments, most or all of the plurality of covalently conjugatedpeptide tags present on the at least one of the plurality ofpeptide-tagged agents may comprise a single type of peptide tag. Incertain further embodiments, most or all of the plurality of covalentlyconjugated peptide tags present on the at least one of the plurality ofpeptide-tagged agents may comprise two, three, or four types of peptidetags. In certain embodiments, most or all of the plurality ofpeptide-tagged agents may comprise a single type of peptide tagcovalently conjugated thereon. In certain embodiments, most or all ofthe plurality of peptide-tagged agents may comprise two, three, or fourtypes of peptide tags covalently conjugated thereon. In certainembodiments, most, for example a preponderance, or substantially all (incertain further embodiments greater than 90%, greater than 95% orgreater than 98% of a certain type of bound peptide tags) of the onetype of the plurality of peptide tags, that are bound, is covalentlyconjugated to only one type of the plurality of peptide-tagged agents.

In certain embodiments, at least one of the at least one of theplurality of peptide-tagged agents may comprise, for example, a captureagent capable of binding with the analyte, for example a monoclonalantibody, an affimer, or an aptamer capable of binding with the analyte.In certain further embodiments, each of the at least one of theplurality of peptide-tagged agents may comprise a single capture agent.In certain embodiments, the plurality of peptide-tagged agents maycomprise a single type of capture agent. In certain embodiments, theplurality of peptide-tagged agents may comprise two, three, or fourtypes of capture agents (and in some embodiments two, three or fourtypes of peptide tags, and some embodiments just a single type ofpeptide tag per type of peptide tag).

In certain embodiments, at least one of the covalently conjugatedpeptide tags may be capable of binding with at least one of theplurality of anti-peptide agents. In certain further embodiments, the atleast one of the plurality of anti-peptide agents may be a singleanti-peptide agent. In certain embodiments, the plurality ofanti-peptide agents may comprise a single type of anti-peptide agent. Incertain embodiments, the plurality of anti-peptide agents may comprisetwo, three, or four types of anti-peptide agents.

In certain embodiments, the permeable zone may further comprise, forexample, a plurality of detectable agents, and optionally the analytecomplex may further comprise, for example, at least one of the pluralityof detectable agents, for example one of the plurality of detectableagents. In certain embodiments, the plurality of detectable agents maycomprise a single type of detectable agent. In certain embodiments, theplurality of detectable agents may comprise two, three, or four types ofdetectable agents. In certain embodiments, the plurality of anti-peptideagents may be present at a ratio of at least 8:1, on a weight:weightbasis, relative to the plurality of detectable agents.

In certain embodiments, the assay platform or components orsub-assemblies thereof may be stored at a predetermined temperature, forexample, at a temperature of 4° C., room temperature, less than 10° C.,less than 20° C., at a temperature of less than 30° C. In certainembodiments, the assay platform or components or sub-assemblies thereofmay be stored prior to use and/or after use at a predeterminedtemperature, for example, at a temperature of 4° C., room temperature,less than 10° C., less than 20° C., or at a temperature of less than 30°C. In certain embodiments, the assay platform may have a shelf-life ofat least two months at the pre-determined temperature, for example ashelf-life of at least 2 months, 6 months, or at least 1 year. Incertain embodiments, the assay platform may have a shelf-life of atleast two months at a pre-determined temperature of storage prior to useof less than 20° C., for example a shelf-life of at least 2 months at apre-determined temperature of storage prior to use of less than 20° C.,6 months at a pre-determined temperature of storage prior to use of lessthan 20° C., or at least 1 year at a pre-determined temperature ofstorage prior to use of less than 30° C.

In certain embodiments, the assay platform or components orsub-assemblies thereof, for example the plurality of peptide-taggedagents and/or the plurality of anti-peptide agents, may be stored underlow light conditions, such as in a sealed container. In certainembodiments, the assay platform or components or sub-assemblies thereof,for example the plurality of peptide-tagged agents and/or the pluralityof anti-peptide agents, may be stored prior to use and/or after useunder low light conditions, such as in a sealed container.

Certain embodiments may provide, for example, an assay platform fordetecting a pre-determined analyte, comprising: i) a permeable zoneconfigured to transport a sample comprising the pre-determined analyte,said permeable zone comprising a plurality of peptide-tagged agents,said plurality of peptide-tagged agents capable of forming an analytecomplex comprising at least one of the plurality of peptide-taggedagents and the pre-determined analyte; and ii) a further permeable zoneconfigured to receive at least a portion of the sample from thepermeable zone, the further permeable zone comprising: a plurality ofanti-peptide agents present at a ratio of at least 100:1, on aweight:weight basis, relative to the plurality of peptide-tagged agents,said plurality of anti-peptide agents configured to immobilize the atleast one of the plurality of peptide-tagged agents. In certainembodiments, the plurality of anti-peptide agents may be configured, forexample, to immobilize the analyte complex.

Certain embodiments may provide a lateral flow device. In certainembodiments, the lateral flow device may comprise, for example, a flowpath that may be defined by, for example, at least a portion of apermeable sub-assembly of the lateral flow device. In certainembodiments, the lateral flow device may further comprise, for example,a release zone that may comprise, for example, a plurality ofpeptide-tagged agents. In certain embodiments, the lateral flow devicemay further comprise, for example, a detection zone that may comprise,for example, a plurality of anti-peptide agents that may be present, forexample, in the flow path at a ratio of at least 100:1, on aweight:weight basis, relative to the plurality of peptide-tagged agents.In certain embodiments, the release zone may be configured, for example,to release at least a portion of said plurality of peptide-tagged agentsinto the flow path. In certain embodiments, at least one of saidplurality of anti-peptide agents may be a binding partner with at leastone of the at least a portion of said plurality of peptide-tagged agentsconfigured to be released.

In certain embodiments, at least a portion of the flow path may becharacterized by passive transport, capillary action, wetting, wicking,or a combination of two or more thereof. In certain embodiments, theplurality of peptide-tagged agents may be bound, releasably bound, orunbound to at least a portion of the permeable sub-assembly. In certainembodiments, at least a portion (for example , most, a preponderance, orsubstantially all (in certain further embodiments greater than 90%,greater than 95% or greater than 98% on a weight:weight basis)) of theplurality of peptide-tagged agents may migrate, dissolve, disconnect,and/or diffuse from at least a portion of the permeable sub-assembly.

In certain embodiments, the plurality of anti-peptide agents may bebound to at least a portion of the permeable sub-assembly. In certainembodiments, the at least a portion of the at least a portion of saidplurality of peptide-tagged agents configured to be released may bebound to at least a further portion of the permeable sub-assembly. Incertain embodiments, said plurality of anti-peptide agents may beconfigured, for example, to immobilize at least a portion of the atleast a portion of said plurality of peptide-tagged agents configured tobe released. In certain further embodiments, at least one of the atleast a portion of said plurality of peptide-tagged agents configured tobe released may be capable of binding (and may, for example, be bound)with an analyte prior to being immobilized by said plurality ofanti-peptide agents. In certain embodiments, at least one of the atleast a portion of the plurality of peptide-tagged agents may comprise,for example, a plurality of covalently conjugated peptide tags, whereinat least one of said covalently conjugated peptide tags may be capableof binding (and may, for example, be bound) with at least one of theplurality of anti-peptide agents. In certain further embodiments, the atleast one of the plurality of anti-peptide agents may be one of theplurality of anti-peptide agents. In certain embodiments, the detectionzone may be positioned in the flow path downstream of the release zone.

In certain embodiments, the release zone may further comprise, forexample, a plurality of detectable agents, and the analyte complex mayfurther comprise, for example, at least one of the plurality ofdetectable agents. In certain further embodiments, the plurality ofdetectable agents may be bound, releasably bound, or unbound to thepermeable sub-assembly. In certain further embodiments, the release zonemay be configured to further release at least a portion of saidplurality of detectable agents into the flow path. In certain furtherembodiments, at least a portion of the plurality of detectable agentsmay be capable of migrating, dissolving, disconnecting, or diffusing(and may, for example, migrate, dissolve, disconnect, or diffuse) fromat least a portion of the permeable sub-assembly.

Certain embodiments may provide, for example, a lateral flow device,comprising: (i) a flow path defined by a permeable sub-assembly of thelateral flow device; (ii) a release zone comprising a plurality ofpeptide-tagged agents, said release zone configured to release at leasta portion of said plurality of peptide-tagged agents into the flow path;and (iii) a detection zone comprising a plurality of anti-peptide agentspresent in the flow path at a ratio of at least 100:1, on aweight:weight basis, relative to the plurality of peptide-tagged agents,wherein at least one of said plurality of anti-peptide agents is abinding partner with at least one of the released at least a portion ofsaid plurality of peptide-tagged agents.

Certain embodiments may provide a lateral flow device. In certainembodiments, the lateral flow device may be configured to detect atleast two analytes, for example at least three analytes, at least fouranalytes, at least five analytes, at least six analytes, at least sevenanalytes, at least eight analytes, at least nine analytes, or configuredto detect at least ten analytes. In certain embodiments, the lateralflow device may be configured to detect up to ten analytes. In certainembodiments, the lateral flow device may comprise, for example, a flowpath that may be defined by, for example, at least a portion of apermeable sub-assembly of the lateral flow device. In certainembodiments, the lateral flow device may further comprise, for example,a release zone that may comprise, for example, a plurality of firstpeptide-tagged agents. In certain embodiments, the lateral flow devicemay further comprise, for example, a detection zone that may comprise,for example, a plurality of first anti-peptide agents that may bepresent, for example, in the flow path at a ratio of at least 100:1, ona weight:weight basis, relative to the plurality of first peptide-taggedagents. In certain embodiments, the release zone may be configured, forexample, to release at least a portion of said plurality of firstpeptide-tagged agents into the flow path. In certain embodiments, the atleast one of said plurality of first anti-peptide agents may beconfigured to immobilize at least one of said plurality of firstpeptide-tagged agents configured to be released. In certain embodiments,the release zone may also comprise, for example, a plurality of secondpeptide-tagged agents. In certain embodiments, the detection zone mayalso comprise, for example, a plurality of second anti-peptide agentsthat may be configured to immobilize at least one of the at least aportion of said plurality of second peptide-tagged agents configured tobe released.

In certain embodiments, the plurality of first peptide-tagged agents maycomprise, for example, a first type of peptide tag, and the plurality ofsecond peptide-tagged agents may comprise, for example, a second type ofpeptide tag. In certain embodiments, the first type of peptide tag andthe second type of peptide tag may be different types of peptide tagsand/or sequences of peptides. In certain embodiments, at least one ofthe plurality of first peptide-tagged agents may comprise, for example,a plurality of covalently conjugated first peptide tags. In certainfurther embodiments, most or all of the plurality of covalentlyconjugated first peptide tags present on the at least one of theplurality of first peptide-tagged agents may comprise a first type ofpeptide tag. In certain embodiments, most or all of the plurality offirst peptide-tagged agents may comprise the first type of peptide tagcovalently conjugated thereon. In certain embodiments, at least one ofthe plurality of second peptide-tagged agents may comprise, for example,a plurality of covalently conjugated second peptide tags. In certainfurther embodiments, most or all of the plurality of covalentlyconjugated second peptide tags present on the at least one of theplurality of second peptide-tagged agents may comprise a second type ofpeptide tag. In certain embodiments, most or all of the plurality ofsecond peptide-tagged agents may comprise the second type of peptide tagcovalently conjugated thereon.

In certain embodiments, at least one of the plurality of firstpeptide-tagged agents may comprise, for example, a first capture agent,for example a first monoclonal antibody, a first affimer, or a firstaptamer, capable of binding with a first analyte. In certain furtherembodiments, each of the at least one of the plurality of firstpeptide-tagged agents may comprise a single first capture agent. Incertain embodiments, the plurality of first peptide-tagged agents maycomprise a single type of first capture agent. In certain embodiments,at least one of the plurality of second peptide-tagged agents maycomprise, for example, a second capture agent, for example a secondmonoclonal antibody, a second affimer, or a second aptamer, capable ofbinding with a second analyte. In certain further embodiments, each ofthe at least one of the plurality of second peptide-tagged agents maycomprise a single second capture agent. In certain embodiments, theplurality of second peptide-tagged agents may comprise a single type ofsecond capture agent.

In certain embodiments, the plurality of first anti-peptide agents maycomprise a first type of anti-peptide agent, and the plurality of secondanti-peptide agents may comprise a second type of anti-peptide agent. Incertain embodiments, the first type of anti-peptide agent and the secondtype of anti-peptide agent may be different types of anti-peptideagents. In certain further embodiments, the first type of anti-peptideagent may be capable of binding with the first type of peptide tag, andthe second type of anti-peptide agent may be capable of binding with thesecond type of peptide tag. In certain further embodiments, the firsttype of anti-peptide agent may be a specific binding partner with thefirst type of peptide tag, and the second type of anti-peptide agent maybe a specific binding partner with the second type of peptide tag.

In certain embodiments, the plurality of first anti-peptide agents maybe interspersed with the plurality of second anti-peptide agents. Incertain embodiments, at least a portion of the plurality of firstanti-peptide agents may be segregated from at least a portion of theplurality of second anti-peptide agents. In certain embodiments, atleast a portion of the plurality of first anti-peptide agents may bedownstream from at least a portion of the plurality of secondanti-peptide agents, relative to the direction of flow of the flow path.In certain embodiments, at least a portion of the plurality of firstanti-peptide agents and at least a portion of the plurality of secondanti-peptide agents may be segregated from one another and positionedside by side, relative to a direction of flow defined by the flow path.

Certain embodiments may provide, for example, a lateral flow device,comprising: (i) a flow path defined by a permeable sub-assembly of thelateral flow device; (ii) a release zone comprising: (a) a plurality offirst peptide-tagged agents, said release zone configured to release atleast a portion of said plurality of first peptide-tagged agents intothe flow path; and (b) a plurality of second peptide-tagged agents, saidrelease zone configured to release at least a portion of said pluralityof second peptide-tagged agents into the flow path; and (iii) adetection zone comprising: (a) a plurality of first anti-peptide agentspresent in the flow path at a ratio of at least 100:1, on aweight:weight basis, relative to the plurality of first peptide-taggedagents, wherein at least one of said plurality of first anti-peptideagents is a binding partner with at least one of the configured to bereleased at least a portion of said plurality of first peptide-taggedagents; and (b) a plurality of second anti-peptide agents present in theflow path at a ratio of at least 100:1, on a weight:weight basis,relative to the plurality of second peptide-tagged agents, wherein atleast one of said plurality of second anti-peptide agents is a bindingpartner with at least one of the configured to be released at least aportion of said plurality of second peptide-tagged agents.

Certain embodiments may provide an assembly. In certain embodiments, theassembly may comprise, for example, a permeable member that may have aplurality of peptide-tagged agents deposited on at least a portionthereof. In certain embodiments, the assembly may further comprise, forexample, a further permeable member that may be in fluid communicationwith the permeable member. In certain embodiments, the plurality ofpeptide-tagged agents may be capable of forming an analyte complex. Incertain embodiments, the analyte complex may comprise, for example, atleast one of the plurality of peptide-tagged agents and an analyte. Incertain embodiments, the further permeable member may have a pluralityof anti-peptide agents bound to at least a portion thereof. In certainembodiments, the plurality of anti-peptide agents may be present, forexample, at a ratio of at least 100:1, on a weight:weight basis,relative to the plurality of peptide-tagged agents. Furthermore, incertain embodiments the plurality of anti-peptide agents may be capableof immobilizing at least one of said at least one of the plurality ofpeptide-tagged agents.

In certain embodiments, fluid communication of the plurality ofpeptide-tagged agents and/or the analyte between the permeable memberand the further permeable member may comprise, for example, passivetransport, capillary action, wetting, wicking, or a combination of twoor more thereof. In certain embodiments, the plurality of peptide-taggedagents may be bound, releasably bound, or unbound to at least a portionof the permeable member. In certain embodiments, at least a portion ofthe plurality of peptide-tagged agents may migrate, dissolve,disconnect, and/or diffuse from at least a portion of the permeablemember. In certain embodiments, at least one of the at least one of theplurality of peptide-tagged agents and at least one of the plurality ofpeptide-tagged agents may be binding partners. In certain embodiments,at least one of the at least one of the plurality of peptide-taggedagents may comprise, for example, a plurality of covalently conjugatedpeptide tags, wherein at least one of said covalently conjugated peptidetags may be capable of binding with at least one of the plurality ofanti-peptide agents. In certain further embodiments, the at least one ofthe plurality of anti-peptide agents may be a single anti-peptide agent.In certain embodiments, the permeable member may be configured, forexample, to fluidly communicate the analyte complex to the furtherpermeable member. In certain embodiments, the permeable member mayfurther comprise, for example, a plurality of detectable agents, andoptionally the analyte complex may further comprise, for example, atleast one of the plurality of detectable agents. In certain furtherembodiments, the plurality of detectable agents may be bound, releasablybound, or unbound to the permeable member. In certain furtherembodiments, at least a portion of the plurality of detectable agentscan migrate, dissolve, disconnect, and/or diffuse from at least aportion of the permeable member.

Certain embodiments may provide, for example, an assembly, comprising:(i) a permeable member having a plurality of peptide-tagged agentsdeposited on at least a portion thereof, said plurality ofpeptide-tagged agents capable of forming an analyte complex, saidanalyte complex comprising at least one of the plurality ofpeptide-tagged agents and a pre-determined analyte; and (ii) a furtherpermeable member in fluid communication with the permeable member, saidfurther permeable member having a plurality of anti-peptide agents boundto at least a portion thereof, said plurality of anti-peptide agentspresent at a ratio of at least 100:1, on a weight:weight basis, relativeto the plurality of peptide-tagged agents, said plurality ofanti-peptide agents capable of immobilizing at least one of said atleast one of the plurality of peptide-tagged agents.

Certain embodiments may utilize the assembly in a method to detect ananalyte present at a low concentration, for example at a concentrationof less than 1 mIU/mL in a sample, or at a concentration of less than 1mM in a sample, or at a concentration of less than 10 ng/mL. In certainembodiments, the analyte concentration may be in the range of 0.001-1mM, for example an analyte concentration in the range of 0.01-1 mM,0.1-1 mM, 0.25-1 mM, or an analyte concentration in the range of0.25-0.75 mM. In certain embodiments, the analyte concentration may bein the range of 0.001-10 ng/mL, for example an analyte concentration inthe range of 0.01-10 ng/mL, 0.1-10 ng/mL, 1-7.5 ng/mL, or an analyteconcentration in the range of 2.5-7.5 ng/m L. In certain embodiments,the method may comprise depositing a sample onto the apparatus. Incertain further embodiments, the method may comprise detecting thetarget analyte by measuring a detection signal having a signal-to-noiseratio of at least 1.67. In certain embodiments, the signal to noiseratio may be defined as the ratio of the detection signal to a detectionsignal obtained if the apparatus is configured and/or used in a similarmanner, for example if the apparatus comprises the same or a similaramount of the plurality of peptide-tagged agent, the the same or asimilar amount of the plurality of anti-peptide agent, and/or the thesame or a similar amount of the plurality of detectable agent, but wherethere is no analyte present.

Certain embodiments may provide an assembly. In certain embodiments, theassembly may comprise, for example, a permeable member that may have aplurality of first peptide-tagged agents deposited on at least a portionthereof. In certain embodiments, the assembly may further comprise, forexample, a further permeable member that may be in fluid communicationwith the permeable member. In certain embodiments, the plurality offirst peptide-tagged agents may be capable of forming a first analytecomplex. In certain embodiments, the first analyte complex may comprise,for example, at least one of the plurality of first peptide-taggedagents and a first analyte. In certain embodiments, the furtherpermeable member may have a plurality of first anti-peptide agents boundto at least a portion thereof. In certain embodiments, the plurality offirst anti-peptide agents may be present, for example, ata ratio of atleast 100:1, on a weight:weight basis, relative to the plurality offirst peptide-tagged agents. In certain further embodiments, theplurality of first anti-peptide agents may be capable of immobilizing atleast one of said at least one of the plurality of first peptide-taggedagents. In certain embodiments, the permeable member may also have, forexample, a plurality of second peptide-tagged agents deposited on atleast a portion thereof. In certain embodiments, the plurality of secondpeptide-tagged agents may be capable of forming a second analytecomplex. In certain embodiments, the second analyte complex maycomprise, for example, at least one of the plurality of secondpeptide-tagged agents and a second analyte. In certain embodiments, thefurther permeable member may also have, for example, a plurality ofsecond anti-peptide agents bound to at least a portion thereof. Incertain embodiments, the plurality of second anti-peptide agents may becapable of immobilizing at least one of said at least one of theplurality of second peptide-tagged agents.

In certain embodiments, the plurality of first peptide-tagged agents andthe plurality of second peptide-tagged agents may be interspersed on thepermeable member. In certain embodiments, the plurality of firstpeptide-tagged agents and the plurality of second peptide-tagged agents,or portions thereof, may be segregated from one another on the permeablemember.

In certain embodiments, the plurality of first peptide-tagged agents andthe plurality of second peptide-tagged agents may comprise two or moredifferent types of peptide-tagged agents, for example two differenttypes of peptide-tagged agents. In certain embodiments, the plurality offirst peptide-tagged agents and the plurality of second peptide-taggedagents may comprise two or more different types of capture agents, forexample two different types of capture agents. In certain embodiments,the plurality of first peptide-tagged agents and the plurality of secondpeptide-tagged agents may comprise two or more different types ofcovalently conjugated peptide tags, for example two different types ofpeptide-tags.

In certain embodiments, the plurality of first anti-peptide agents andthe plurality of second anti-peptide agents may comprise two or moredifferent types of anti-peptide agents, for example two different typesof anti-peptide agents. In certain embodiments, the plurality of firstanti-peptide agents and the plurality of second anti-peptide agents mayhave one or more type of anti-peptide agents in common, for example onetype of anti-peptide agent in common.

In certain embodiments, the plurality of first anti-peptide agents andthe plurality of second anti-peptide agents may be interspersed on thepermeable member. In certain embodiments, the plurality of firstanti-peptide agents and the plurality of second anti-peptide agents, orportions thereof, may be segregated from one another on the permeablemember.

In certain embodiments, at least a portion of the plurality of firstanti-peptide agents may be bound in a first sub-zone of the detectionzone. In certain embodiments, at least a portion of the plurality ofsecond anti-peptide agents may be bound in a second sub-zone of thedetection zone. In certain embodiments, the first sub-zone and thesecond sub-zone may be overlapping. In certain embodiments, the firstsub-zone and the second sub-zone may be disjoint. In certainembodiments, a portion of the sample, or a portion of the portion of thesample, may migrate through the first sub-zone before said portionmigrates through the second sub-zone.

Certain embodiments may provide, for example, an assembly, comprising:(i)a permeable member having: (a) a plurality of first peptide-taggedagents deposited on at least a portion thereof, said plurality of firstpeptide-tagged agents capable of forming a first analyte complex, saidfirst analyte complex comprising at least one of the plurality of firstpeptide-tagged agents and a first pre-determined analyte; and (b) aplurality of second peptide-tagged agents deposited on at least aportion thereof, said plurality of second peptide-tagged agents capableof forming a second analyte complex, said second analyte complexcomprising at least one of the plurality of second peptide-tagged agentsand a second pre-determined analyte; and (ii) a further permeable memberin fluid communication with the permeable member, said further permeablemember having: (a) a plurality of first anti-peptide agents bound to atleast a portion thereof, said plurality of first anti-peptide agentspresent at a ratio of at least 100:1, on a weight:weight basis, relativeto the plurality of first peptide-tagged agents, said plurality of firstanti-peptide agents capable of immobilizing at least one of the said atleast one of the plurality of first peptide-tagged agents; and (b) aplurality of second anti-peptide agents bound to at least a portionthereof, said plurality of second anti-peptide agents present at a ratioof at least 100:1, on a weight:weight basis, relative to the pluralityof second peptide-tagged agents, said plurality of second anti-peptideagents capable of immobilizing at least one of the said at least one ofthe plurality of second peptide-tagged agents.

Certain embodiments may provide a permeable assembly. In certainembodiments, the permeable assembly may comprise, for example, a flowpath defined by, for example, the permeable assembly. In certainembodiments, the permeable assembly may further comprise, for example, asample zone. In certain embodiments, the permeable assembly may furthercomprise, for example, a detection zone. In certain embodiments, thesample zone may comprise, for example, a plurality of analyte complexes.In certain further embodiments, said plurality of analyte complexes maycomprise, for example, a plurality of peptide-tagged agents. In certainembodiments, the detection zone may comprise, for example, a pluralityof anti-peptide agents present, for example, in the flow path at a ratioof at least 100:1, on a weight:weight basis, relative to the pluralityof peptide-tagged agents. In certain embodiments, the plurality ofanti-peptide agents may be capable of immobilizing at least a portion ofthe plurality of peptide-tagged agents.

In certain embodiments, the sample zone may be configured, for example,to release at least a portion of said plurality of analyte complexesinto the flow path. In certain embodiments, the plurality ofanti-peptide agents may be positioned in the flow path downstream of thesample zone. In certain embodiments, the sample zone and the detectionzone may be in spatial relation to one another, for example abutting,adjacent, and/or collinear to one another. In certain embodiments, thedetection zone may be housed, for example, in a casing. In certainfurther embodiments, the casing may define, for example, a viewing hole,a window, and/or a transparent section positioned proximate thedetection zone. In certain further embodiments, the sample zone may behoused, for example, in the casing. In certain further embodiments, thesample zone may be configured, for example, to receive the plurality ofanalytes through a sample inlet defined by, for example, the casing.

Certain embodiments may provide, for example, a permeable assembly,comprising: (i) a flow path defined by the permeable assembly; (ii) asample zone, comprising: a plurality of analyte complexes, saidplurality of analyte complexes comprising a plurality of peptide-taggedagents; and (iii) a detection zone, comprising: a plurality ofanti-peptide agents present in the flow path at a ratio of at least100:1, on a weight:weight basis, relative to the plurality ofpeptide-tagged agents, said plurality of anti-peptide agents capable ofimmobilizing at least a portion of the plurality of peptide-taggedagents.

Certain embodiments may provide an assembly. In certain embodiments, theassembly may comprise, for example, a permeable member having aplurality of analyte complexes deposited on at least a portion thereof.In certain embodiments, the assembly may further comprise, for example,a further permeable member that may be configured, for example, toreceive at least a portion of the analyte complexes from the permeablemember. In certain embodiments, the plurality of analyte complexes maycomprise, for example, a plurality of peptide-tagged agents. In certainembodiments, the further permeable member may have a plurality ofanti-peptide agents bound to at least a portion thereof. In certainembodiments, the plurality of anti-peptide agents may be present, forexample, at a ratio of at least 100:1, on a weight:weight basis,relative to the plurality of peptide-tagged agents. Furthermore, incertain embodiments the plurality of anti-peptide agents may beconfigured, for example, to immobilize said at least one of theplurality of peptide-tagged agents to the permeable assembly.

In certain embodiments, the permeable member and the further permeablemember may be in spatial relation to one another, for example abutting,adjacent, and/or collinear to one another. In certain embodiments, thepermeable member may comprise, for example, an absorbent pad. In certainembodiments, the permeable member may be housed, for example, in acasing. In certain further embodiments, the permeable member may beconfigured, for example, to receive the plurality of analytes through asample inlet defined by, for example, the casing. In certainembodiments, the further permeable member may comprise, for example, atleast one porous membrane. In certain embodiments, the further permeablemember may be housed, for example, in the casing. In certain furtherembodiments, the casing defines, for example, a viewing hole, window,and/or a transparent section positioned proximate the further permeablemember. In certain embodiments, the assembly may further may comprise,for example, an absorbent member positioned in the flow path downstreamof the further permeable member.

Certain embodiments may provide, for example, an assembly, comprising:(i) a permeable member having a plurality of analyte complexes depositedon at least a portion thereof, said permeable member configured totransport at least a portion of the analyte complexes, said plurality ofanalyte complexes comprising a plurality of peptide-tagged agents; and(ii) a further permeable member configured to receive at least theportion of the analyte complexes from the permeable member, said furtherpermeable member having a plurality of anti-peptide agents bound to atleast a portion thereof, said plurality of anti-peptide agents: (a)present at a ratio of at least 100:1, on a weight:weight basis, relativeto the plurality of peptide-tagged agents; and (b) configured toimmobilize said at least one of the plurality of peptide-tagged agentsto the permeable assembly.

Certain embodiments may provide a lateral flow device for detecting ananalyte. In certain embodiments, the lateral flow device may comprise,for example, a flow path defined by, for example, a permeablesub-assembly of the lateral flow device. In certain embodiments, thelateral flow device may further comprise, for example, a sample zonethat may be configured, for example, to receive the analyte and torelease the analyte into the flow path. In certain embodiments, thelateral flow device may further comprise, for example, a release zonethat may comprise a plurality of peptide-tagged agents. In certainembodiments, the lateral flow device may further comprise, for example,a detection zone that may comprise a plurality of anti-peptide agentspresent, for example, in the flow path at a ratio of at least 100:1, ona weight:weight basis, relative to the plurality of peptide-taggedagents. In certain embodiments, the release zone may be configured, forexample, to release at least a portion of said plurality ofpeptide-tagged agents into the flow path. In certain embodiments, atleast one of said plurality of anti-peptide agents may be a bindingpartner with at least one of the at least a portion of said plurality ofpeptide-tagged agents configured to be released.

In certain embodiments, the release zone may be positioned in the flowpath downstream of the sample zone. In certain embodiments, thedetection zone may be positioned in the flow path downstream of therelease zone. In certain embodiments, the sample zone and the releasezone may be in spatial relation to one another, for example abutting,adjacent, and/or collinear to one another. In certain embodiments, therelease zone and the detection zone may be in spatial relation to oneanother, for example abutting, adjacent, and/or collinear to oneanother. In certain embodiments, the sample zone and the detection zonemay not be in spatial relation to one another, for example not abutting,not adjacent, and not collinear to one another. In certain embodiments,the release zone and the detection zone may be housed, for example, in acasing. In certain further embodiments, the sample zone may be housed,for example, in the casing. In certain further embodiments, the samplezone may be configured, for example, to receive the analyte through asample inlet defined by, for example, the casing. In certainembodiments, the lateral flow device may further comprise, for example,a plurality of detectable agents. In certain further embodiments, theplurality of detectable agents may be introduced to the permeablesub-assembly separately from the analyte. In certain furtherembodiments, the plurality of detectable agents may be bound to thepermeable sub-assembly upstream of, downstream of, side by side with, orinterspersed with said plurality of peptide-tagged agents.

Certain embodiments may provide, for example, a lateral flow device fordetecting a pre-determined analyte, comprising: (i) a flow path definedby a permeable sub-assembly of the lateral flow device; (ii) a samplezone configured to receive the pre-determined analyte and to release thepre-determined analyte into the flow path; (iii) a release zonecomprising a plurality of peptide-tagged agents, said release zoneconfigured to release at least a portion of said plurality ofpeptide-tagged agents into the flow path; and (iv) a detection zonecomprising a plurality of anti-peptide agents present in the flow pathat a ratio of at least 100:1, on a weight:weight basis, relative to theplurality of peptide-tagged agents, wherein at least one of saidplurality of anti-peptide agents is a binding partner with at least oneof the released at least a portion of said plurality of peptide-taggedagents.

Certain embodiments may provide a lateral flow device. In certainembodiments, the lateral flow device may comprise, for example, a firstpermeable member that may be configured, for example, to receive asample containing an analyte and, optionally, to transport said analyte.In certain embodiments, the lateral flow device may further comprise asecond permeable member that may be configured, for example, to receivethe analyte from the first member. In certain embodiments, the secondpermeable member may have a plurality of peptide-tagged agents depositedon at least a portion thereof. In certain embodiments, the plurality ofpeptide-tagged agents may be capable of forming an analyte complex. Incertain embodiments, the analyte complex may comprise, for example, atleast one of the plurality of peptide-tagged agents and the analyte. Incertain embodiments, the lateral flow device may further comprise, forexample, a third permeable member that may be configured, for example,to receive the analyte complex from the second permeable member. Incertain embodiments, the third permeable member may have a plurality ofanti-peptide agents bound to at least a portion thereof. In certainembodiments, the plurality of anti-peptide agents may be present, forexample, at a ratio of at least 100:1, on a weight:weight basis,relative to the plurality of peptide-tagged agents. Furthermore, incertain embodiments, the plurality of anti-peptide agents may beconfigured, for example, to immobilize at least one of said at least oneof the plurality of peptide-tagged agents.

In certain embodiments, the first permeable member and the secondpermeable member may be in spatial relation to one another, for exampleabutting, adjacent, and/or collinear to one another. In certainembodiments, the second permeable member and the third permeable membermay be in spatial relation to one another, for example abutting,adjacent, and/or collinear to one another. In certain embodiments, thefirst permeable member and the third permeable member may not be inspatial relation to one another, for example not abutting, not adjacent,and not collinear to one another. In certain embodiments, secondpermeable member and the third permeable member may be housed, forexample, in a casing. In certain further embodiments, the firstpermeable member may be housed, for example, in the casing. In certainfurther embodiments, the first permeable member may be configured, forexample, to receive the sample through a sample inlet defined by, forexample, the casing. In certain embodiments, the lateral flow device mayfurther comprise, for example, a plurality of detectable agents. Incertain further embodiments, the analyte complex may further comprise,for example, the analyte bound to one or more of the plurality ofdetectable agents, for example one of the plurality of detectableagents. In certain further embodiments, the plurality of detectableagents may be introduced to the lateral flow device separately from thesample. In certain further embodiments, the plurality of detectableagents may be bound to the second permeable member upstream of,downstream of, side by side with, or interspersed with said plurality ofpeptide-tagged agents. In certain embodiments, the lateral flow devicemay further comprise, for example, an absorbent member positioned in theflow path downstream of the first, second, and third permeable members.In certain further embodiments, the absorbency of the absorbent membermay exceed the absorbency of the first permeable member, for example thequantity of a liquid which may be absorbed by the absorbent member mayexceed that of the first permeable member by a factor of at least 2, forexample at least 5, 10, 25, 50, or at least 100.

Certain embodiments may provide, for example, a lateral flow device,comprising: (i) a first permeable member configured to receive a samplecontaining a pre-determined analyte and to transport said pre-determinedanalyte; (ii) a second permeable member configured to receive thepre-determined analyte from the first member, said second permeablemember having a plurality of peptide-tagged agents deposited on at leasta portion thereof, said plurality of peptide-tagged agents capable offorming an analyte complex, said analyte complex comprising at least oneof the plurality of peptide-tagged agents and the pre-determinedanalyte; and (iii) a third permeable member configured to receive theanalyte complex from the second permeable member, said third permeablemember having a plurality of anti-peptide agents bound to at least aportion thereof, said plurality of anti-peptide agents: (a) present at aratio of at least 100:1, on a weight:weight basis, relative to theplurality of peptide-tagged agents; and (b) configured to immobilize atleast one of the said at least one of the plurality of peptide-taggedagents.

Certain embodiments may provide a lateral flow device for detecting ananalyte. In certain embodiments, the lateral flow device may comprise,for example, a flow path defined by, for example, a permeablesub-assembly of the lateral flow device. In certain embodiments, thelateral flow device may further comprise a solvent zone that may beconfigured, for example, to receive a solvent and, optionally, tocommunicate a portion of said solvent into the flow path. In certainembodiments, the lateral flow device may be configured, for example, toreceive a sample in a sample zone of the lateral flow device and,optionally, to introduce at least a portion of said sample into the flowpath. In certain embodiments, the lateral flow device may furthercomprise a release zone that may comprise a plurality of peptide-taggedagents. In certain embodiments, the lateral flow device may furthercomprise a detection zone that may comprise a plurality of anti-peptideagents present, for example, in the flow path at a ratio of at least100:1, on a weight:weight basis, relative to the plurality ofpeptide-tagged agents. In certain embodiments, the at least a portion ofsaid sample may contain the analyte. In certain embodiments, the releasezone may be configured, for example, to release at least a portion ofsaid plurality of peptide-tagged agents into the flow path. In certainembodiments, the plurality of anti-peptide agents may be configured, forexample, to immobilize at least a fraction of the at least a portion ofsaid plurality of peptide-tagged agents configured to be released. Incertain embodiments, the sample zone may be positioned in the flow pathdownstream of the release zone.

In certain embodiments, the release zone may be positioned in the flowpath downstream of the sample zone. In certain embodiments, the samplemay be a solid, a gel, a dense sample, and/or a congealed sample. Incertain embodiments, the sample may dissolve, mix, passively mix, and/ordiffuse into the solvent. In certain embodiments, the analyte maymigrate, dissolve, disconnect, and/or diffuse from the sample into thesolvent. In certain embodiments, the release zone may be housed, forexample, in a casing. In certain further embodiments, the sample zonemay be housed, for example, in a casing. In certain further embodiments,the sample may be introduced to the lateral flow device through a sampleinlet defined by, for example, the casing. In certain embodiments, thesolvent zone may be configured, for example, to receive the solventthrough a solvent inlet defined by, for example, the casing. In certainfurther embodiments, the sample inlet may be shaped to allow the sampleto be spread, pipetted, and/or smeared onto the sample zone.

Certain embodiments may provide, for example, a lateral flow device fordetecting a pre-determined analyte, comprising: (i) a flow path definedby a permeable sub-assembly of the lateral flow device; (ii) a solventzone configured to receive a solvent and to communicate a portion ofsaid solvent into the flow path; (iii) a sample zone configured toreceive a sample and to introduce at least a portion of said sample intothe flow path, said at least a portion of said sample containing thepre-determined analyte; (iv) a release zone comprising a plurality ofpeptide-tagged agents, said release zone configured to release at leasta portion of said plurality of peptide-tagged agents into the flow path;and (v) a detection zone comprising a plurality of anti-peptide agentspresent in the flow path at a ratio of at least 100:1, on aweight:weight basis, relative to the plurality of peptide-tagged agents,said plurality of anti-peptide agents configured to immobilize at leasta portion of the released at least a portion of said plurality ofpeptide-tagged agents.

Certain embodiments may provide a lateral flow device. In certainembodiments, the lateral flow device may comprise, for example, a firstpermeable member that may be configured, for example, to receive asolvent. In certain embodiments, the lateral flow device may furthercomprise, for example, a second permeable member that may be in fluidcommunication with the first permeable member. In certain embodiments,the lateral flow device may further comprise, for example, a thirdpermeable member that may be in fluid communication with the firstpermeable member. In certain embodiments, the lateral flow device mayfurther comprise, for example, a fourth permeable member that may be influid communication with at least the second and the third permeablemembers. In certain embodiments, the second permeable member may beconfigured, for example, to receive a sample containing an analyte. Incertain embodiments, the third permeable member may have a plurality ofpeptide-tagged agents deposited thereon. In certain embodiments, thefourth permeable member may have a plurality of anti-peptide agentsbound thereto at a ratio of at least 100:1, on a weight:weight basis,relative to the plurality of peptide-tagged agents.

Certain embodiments may provide, for example, a lateral flow device,comprising: (i) a first permeable member configured to receive asolvent; (ii) a second permeable member in fluid communication with thefirst permeable member, said second permeable member configured toreceive a sample containing a pre-determined analyte; (iii) a thirdpermeable member in fluid communication with the first permeable member,said third permeable member having a plurality of peptide-tagged agentsdeposited thereon; and (iv) a fourth permeable member in fluidcommunication with at least the second and the third permeable members,said fourth permeable member having a plurality of anti-peptide agentsbound thereto at a ratio of at least 100:1, on a weight:weight basis,relative to the plurality of peptide-tagged agents.

Certain embodiments provide lateral flow methods, including, forexample, methods of detection, methods of making lateral flowapparatuses, and methods of making lateral flow kits. Other types oflateral flow methods are contemplated herein.

Certain embodiments may provide a method. In certain embodiments, themethod may comprise, for example, exposing a sample that may comprise ananalyte to a plurality of peptide-tagged agents to form an analytecomplex. In certain further embodiments, the method may comprise, forexample, exposing the analyte complex to a plurality of detectableagents, to result in an analyte complex comprising at least one of theplurality of detectable agents. In certain embodiments, the method maycomprise, for example, exposing a sample that may comprise an analyte toa plurality of detectable agents to form an analyte complex. In certainfurther embodiments, the method may comprise, for example, exposing theanalyte complex to a plurality of peptide-tagged agents, to result in ananalyte complex comprising at least one of the plurality ofpeptide-tagged agents. In certain embodiments, the method may furthercomprise, for example, transporting the analyte complex to a detectionzone of a lateral flow device. In certain embodiments, the analytecomplex may comprise, for example, the analyte and at least one of theplurality of peptide-tagged agents. In certain embodiments, thedetection zone may comprise, for example, a plurality of anti-peptideagents present, for example, at a ratio of at least 100:1, on aweight:weight basis, relative to the plurality of peptide-tagged agents.In certain embodiments, the plurality of anti-peptide agents may becapable of immobilizing at least one of the at least one of theplurality of peptide-tagged agents.

In certain embodiments, transporting may comprise, for example, bulktransport, passive transport, capillary action, wetting, wicking, or acombination of two or more thereof on a lateral flow device. In certainembodiments, the at least one of the at least one of the plurality ofpeptide-tagged agents may comprise, for example, a plurality ofcovalently conjugated peptide tags, wherein at least one of saidcovalently conjugated peptide tags may be configured, for example, tobind with at least one of the plurality of anti-peptide agents. Incertain embodiments, the analyte complex may be formed on a portion ofthe lateral flow device. In certain embodiments, at least a portion ofthe analyte complex may not be formed on any portion of the lateral flowdevice. In certain embodiments, the method may further comprise, forexample, releasing said plurality of peptide-tagged agents from a boundstate on a portion of the lateral flow device prior to forming theanalyte complex, for example by exposing said plurality ofpeptide-tagged agents to a sample and/or solvent. In certainembodiments, the method may further comprise, for example, introducingsaid plurality of peptide-tagged agents in a liquid to the lateral flowdevice. In certain embodiments, forming the analyte complex may furthercomprise, for example, exposing at least a portion of sample and atleast a portion of the plurality of peptide-tagged agents to a solvent.In certain further embodiments, the at least a portion of the sample maybe exposed to the solvent before, after, or coincident with the at leasta portion of the plurality of peptide-tagged agents. In certainembodiments, the method may further comprise, for example, immobilizingthe analyte complex to a portion of the lateral flow device. In certainembodiments, the method may further comprise, for example, immobilizingthe analyte complex to a portion of the detection zone. In certainembodiments, the method may further comprise, for example, binding atleast one of the at least one of the plurality of peptide-tagged agentsto at least one of the plurality of anti-peptide agents. In certainfurther embodiments, the method may further comprise, for example,releasing said plurality of detectable agents from a bound state on aportion of the lateral flow device, for example by exposing saidplurality of detectable agents to a sample and/or solvent. In certainother embodiments, the method may further comprise, for example,introducing said plurality of detectable agents in a liquid to thelateral flow device. In certain embodiments, the method may furthercomprise, for example, detecting said immobilized analyte complex. Incertain further embodiments, the method may comprise, for example,detecting said immobilized analyte complex by detecting a detectableagent bound to the immobilized analyte complex.

In certain embodiments, the detecting may be accomplished in less than 2hours, for example in less than 90 minutes, 80 minutes, 70 minutes, 60minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 18 minutes, 16minutes, 14 minutes, 12 minutes, 10 minutes, 9 minutes, 8 minutes, 7minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or inless than 1 minute. In certain embodiments, the detecting may beaccomplished in the range of between 1 minute and 20 minutes, forexample in the range of between 1 minute and 5 minutes, 5 minutes and 15minutes, 5 minutes and 10 minutes, or in the range of between 3 minutesand 8 minutes. In certain embodiments, the detecting may require atleast 10 minutes, for example at least 15 minutes, 20 minutes, 30minutes, 45 minutes, or at least 1 hour.

Certain embodiments may provide, for example, a method, comprising: (i)exposing a sample comprising a pre-determined analyte to a plurality ofpeptide-tagged agents to form an analyte complex, said analyte complexcomprising the pre-determined analyte and at least one of the pluralityof peptide-tagged agents; and (ii) transporting the analyte complex to adetection zone of a lateral flow device, said detection zone comprisinga plurality of anti-peptide agents present at a ratio of at least 100:1,on a weight:weight basis, relative to the plurality of peptide-taggedagents, said plurality of anti-peptide agents capable of immobilizing atleast one of the at least one of the plurality of peptide-tagged agents.

Certain embodiments may provide a method. In certain embodiments, themethod may be configured to detect at least two analytes, for example atleast two analytes, at least three analytes, at least four analytes, atleast five analytes, at least six analytes, at least seven analytes, atleast eight analytes, at least nine analytes, or detecting at least tenanalytes. In certain embodiments, the method may be configured to detectup to ten analytes. In certain embodiments, the method may comprise, forexample, exposing a sample that may comprise a first analyte and asecond analyte to a plurality of peptide-tagged agents to form at leasta first analyte complex and a second analyte complex. In certainembodiments, the method may further comprise, for example, transportingat least the first analyte complex and the second analyte complex to adetection zone of a lateral flow device. In certain embodiments, thefirst analyte complex may comprise, for example, the first analyte andat least a first one of the plurality of peptide-tagged agents. Incertain embodiments, the second analyte complex may comprise, forexample, the second analyte and at least a second one of the pluralityof peptide-tagged agents. In certain embodiments, the detection zone maycomprise, for example, a plurality of anti-peptide agents present, forexample, at a ratio of at least 100:1, on a weight:weight basis,relative to the plurality of peptide-tagged agents. In certainembodiments, the plurality of anti-peptide agents may be capable ofimmobilizing at least one of the at least one of the plurality ofpeptide-tagged agents. In certain embodiments, the plurality ofanti-peptide agents may be configured to immobilize the first analytecomplex and the second analyte complex.

In certain embodiments, the first one of the plurality of peptide-taggedagents may comprise a first type of peptide tag and a first type ofcapture agent. In certain embodiments, the second one of the pluralityof peptide-tagged agents may comprise a second type of peptide tag and asecond type of capture agent. In certain further embodiments, the firsttype of peptide tag and the second type of peptide tag may be differenttypes of peptide tags and/or sequences of peptides. In certain furtherembodiments, the first type of capture agent and the second type ofcapture agent may be different types of capture agents, for exampledifferent types of antibodies. In certain embodiments, the plurality ofanti-peptide agents may comprise at least two types of anti-peptideagents. In certain further embodiments, the plurality of anti-peptideagents may comprise a first type of anti-peptide agent which may beconfigured to bind with the first type of peptide tag, and the pluralityof anti-peptide agents may further comprise a second type ofanti-peptide agent which may be configured to bind with the second typeof peptide tag. In certain further embodiments, the first type ofanti-peptide agent may be a specific binding partner with the first typeof peptide tag, and the second type of anti-peptide agent may be aspecific binding partner with the second type of peptide tag.

Certain embodiments may provide, for example, a method of detecting atleast two analytes, comprising: (i) exposing a sample comprising a firstanalyte and a second analyte to a plurality of peptide-tagged agents toform a first analyte complex and a second analyte complex, (a) saidfirst analyte complex comprising the first analyte and at least a firstone of the plurality of peptide-tagged agents; and (b) said secondanalyte complex comprising the second analyte and at least a second oneof the plurality of peptide-tagged agents; and (ii) transporting thefirst analyte complex and the second analyte complex to a detection zoneof a lateral flow device, said detection zone comprising a plurality ofanti-peptide agents present ata ratio of at least 100:1, on aweight:weight basis, relative to the plurality of peptide-tagged agents,said plurality of anti-peptide agents configured to immobilize the firstanalyte complex and the second analyte complex.

Certain embodiments, for example, may provide a method. In certainembodiments, the method may comprise, for example, exposing a samplethat may comprise a first analyte and a second analyte to a plurality offirst peptide-tagged agents and to a plurality of second peptide-taggedagents to form a first analyte complex and a second analyte complex. Incertain embodiments, the method may further comprise, for example,transporting the first analyte complex and the second analyte complex toa detection zone of a lateral flow device. In certain embodiments, thefirst analyte complex may comprise, for example, the first analyte andat least one of the plurality of first peptide-tagged agents. In certainembodiments, the second analyte complex may comprise, for example, thesecond analyte and at least one of the plurality of secondpeptide-tagged agents. In certain embodiments, for example, thedetection zone may comprise, for example, a plurality of firstanti-peptide agents present, for example, at a ratio of at least 100:1,on a weight:weight basis, relative to the plurality of firstpeptide-tagged agents. In certain further embodiments, for example, thedetection zone may comprise, for example, a plurality of secondanti-peptide agents present, for example, at a ratio of at least 100:1,on a weight:weight basis, relative to the plurality of secondpeptide-tagged agents. In certain embodiments, the plurality of firstanti-peptide agents may be configured to immobilize the first analytecomplex. In certain embodiments, the plurality of second anti-peptideagents may be configured to immobilize the second analyte complex.

In certain embodiments, at least a portion of the plurality of firstanti-peptide agents may be bound in a first sub-zone of the detectionzone. In certain embodiments, at least a portion of the plurality ofsecond anti-peptide agents may be bound in a second sub-zone of thedetection zone. In certain embodiments, the first sub-zone and thesecond sub-zone may be overlapping. In certain embodiments, the firstsub-zone and the second sub-zone may be disjoint. In certainembodiments, a portion of the sample may migrate through the firstsub-zone before at least a portion of said portion migrates through thesecond sub-zone.

Certain embodiments may provide, for example, a method of detecting atleast two analytes, comprising: (i) exposing a sample comprising a firstanalyte and a second analyte to a plurality of first peptide-taggedagents and to a plurality of second peptide-tagged agents to form afirst analyte complex and a second analyte complex, (a) said firstanalyte complex comprising the first analyte and at least one of theplurality of first peptide-tagged agents; and (b) said second analytecomplex comprising the second analyte and at least one of the pluralityof second peptide-tagged agents; and (ii) transporting the first analytecomplex and the second analyte complex to a detection zone of a lateralflow device, said detection zone comprising: (a) a plurality of firstanti-peptide agents present at a ratio of at least 100:1, on aweight:weight basis, relative to the plurality of first peptide-taggedagents, said plurality of first anti-peptide agents configured toimmobilize the first analyte complex; and (b) a plurality of secondanti-peptide agents configured to immobilize the second analyte complex.

Certain embodiments may provide a multi-purpose lateral flow test kit.In certain embodiments, the multi-purpose lateral flow kit may comprise,for example, a universal receiver, wherein the universal receiver may beconfigured, for example, to receive any one of a plurality of permeablemembers and, optionally, to bring said any one of the plurality ofpermeable members into fluid communication with a detection zone. Incertain embodiments, the detection zone may comprise, for example, atleast one anti-peptide agent. In certain embodiments, the multi-purposelateral flow kit may further comprise a first permeable member of theplurality of permeable members. In certain embodiments, themulti-purpose lateral flow kit may further comprise a second permeablemember of the plurality of permeable members. In certain embodiments,the first permeable member may comprise, for example, a firstpeptide-tagged agent that may be capable of binding with a firstpre-determined type of analyte. In certain embodiments, the secondpermeable member may comprise, for example, a second peptide-taggedagent that may be capable of binding with a second pre-determined typeof analyte. In certain embodiments, each of the first and secondpeptide-tagged agents may be a binding parter with at least one of theat least one anti-peptide agent.

Certain embodiments may provide, for example, a multi-purpose lateralflow test kit, comprising: (i)a universal receiver, said universalreceiver configured to receive any one of a plurality of permeablemembers and to bring said any one of the plurality of permeable membersinto fluid communication with a detection zone, said detection zonecomprising at least one anti-peptide agent; (ii) a first permeablemember of the plurality of permeable members, comprising a firstpeptide-tagged agent capable of binding with a first pre-determinedanalyte; and (iii) a second permeable member of the plurality ofpermeable members, comprising a second peptide-tagged agent capable ofbinding with a second pre-determined analyte, wherein each of the firstand second peptide-tagged agents is a binding partner with at least oneof the at least one anti-peptide agent.

Certain embodiments may provide a divergent flow device for detecting ananalyte. In certain embodiments, the divergent flow device may comprisea plurality of peptide-tagged agents. In certain embodiments, thedivergent flow device may comprise a central release zone that may beconfigured to introduce said plurality of peptide-tagged agents into adivergent flow path. In certain embodiments, a plurality of anti-peptideagents may be present at a ratio of at least 100:1, on a weight:weightbasis, relative to the plurality of peptide-tagged agents. In certainembodiments, the plurality of anti-peptide agents may be positioned inthe divergent flow path about the central release zone. In certainembodiments, the divergent flow device may be configured to immobilizeat least one of the plurality of peptide-tagged agents. In certainembodiments, the divergent flow path may be a radial flow path.

Certain embodiments may provide, for example, a divergent flow device,comprising: (i) a plurality of peptide-tagged agents; (ii) a centralrelease zone configured to introduce said plurality of peptide-taggedagents into a divergent flow path; and (iii) a plurality of anti-peptideagents present at a ratio of at least 100:1, on a weight:weight basis,relative to the plurality of peptide-tagged agents, said plurality ofanti-peptide agents: (a) positioned in the divergent flow path about thecentral release zone; and (b) capable of immobilizing at least one ofthe plurality of peptide-tagged agents.

Certain embodiments may provide a divergent flow device. In certainembodiments, the divergent flow device may be configured to detect aplurality of different types of analytes, for example at least twodifferent types of analytes, at least three different types of analytes,at least four different types of analytes, at least five different typesof analytes, at least six different types of analytes, at least sevendifferent types of analytes, at least eight different types of analytes,at least nine different types of analytes, or configured to detect atleast ten different types of analytes. In certain embodiments, thedivergent flow device may be configured to detect up to ten differenttypes of analytes. In certain embodiments, the divergent flow device maycomprise a plurality of peptide-tagged agents.

In certain embodiments, the divergent flow device may comprise a centralsample zone that may be configured to communicate a sample containing aplurality of analytes into a divergent flow path. In certain furtherembodiments, the divergent flow path may comprise a first sub-path. Incertain embodiments, the divergent flow path may further comprise asecond sub-path. In certain embodiments, the first sub-path may comprisea plurality of first peptide-tagged agents, at least a portion of saidplurality of first peptide-tagged agents capable of selectively bindingwith a first type of analyte. In certain further embodiments, the firstsubpath may further comprise a plurality of first anti-peptide agents,at least a portion of said plurality of first anti-peptide agentscapable of selectively binding with at least a portion of said pluralityof first peptide-tagged agents. In certain further embodiments, theplurality of first anti-peptide agents may be present at a ratio of atleast 100:1, on a weight:weight basis, relative to the plurality offirst peptide-tagged agents. In certain further embodiments, theplurality of first anti-peptide agents may be configured to immobilizeat least one of the plurality of first peptide-tagged agents. In certainembodiments, the second sub-path may comprise a plurality of secondpeptide-tagged agents, at least a portion of said plurality of secondpeptide-tagged agents capable of selectively binding with a second typeof analyte. In certain further embodiments, the second subpath mayfurther comprise a plurality of second anti-peptide agents, at least aportion of said plurality of second anti-peptide agents capable ofselectively binding with at least a portion of said plurality of secondpeptide-tagged agents. In certain further embodiments, the plurality ofsecond anti-peptide agents may be present at a ratio of at least 100:1,on a weight:weight basis, relative to the plurality of secondpeptide-tagged agents. In certain further embodiments, the plurality ofsecond anti-peptide agents may be configured to immobilize at least oneof the plurality of second peptide-tagged agents.

In certain embodiments, the divergent flow path may flow in a radialdirection defined by the divergent flow device. In certain furtherembodiments, the first sub-path may be defined by a section of thedivergent flow path. In certain further embodiments, the second sub-pathmay be defined by a further section of the divergent flow path. Incertain embodiments, the divergent flow path may be defined by apermeable sub-assembly of the of the divergent flow device.

In certain embodiments, the first sub-path and the second sub-path arein fluid communication. In certain embodiments, the first sub-path andthe second sub-path are not in fluid communication.

Certain embodiments may provide, for example, a divergent flow devicefor detecting a plurality of analytes, comprising: a central sample zoneconfigured to communicate a sample containing a plurality of analytesinto a divergent flow path, said divergent flow path comprising: (i) afirst sub-path comprising: (a) a plurality of first peptide-taggedagents; and (b) a first plurality of anti-peptide agents present at aratio of at least 100:1, on a weight:weight basis, relative to theplurality of first peptide-tagged agents, said plurality of firstanti-peptide agents capable of immobilizing at least one of theplurality of first peptide-tagged agents; and (ii) a second sub-pathcomprising: (a) a plurality of second peptide-tagged agents; and (b)said second sub-path comprising a second plurality of anti-peptideagents capable of immobilizing at least one of the plurality of secondpeptide-tagged agents.

Certain embodiments may provide a divergent flow device for detecting aplurality of analytes. In certain embodiments, the divergent flow devicemay comprise a central sample zone that may be configured to release asample containing the plurality of analytes into a first divergent flowpath and a second divergent flow path. In certain embodiments, thedivergent flow device may further comprise a first release zone in thefirst divergent flow path that may be configured to release a pluralityof first peptide-tagged agents into the first divergent flow path. Incertain embodiments, the divergent flow device may further comprise asecond release zone in the second divergent flow path that may beconfigured to release a plurality of second peptide-tagged agents intothe second divergent flow path. In certain embodiments, the divergentflow device may further comprise a plurality of first anti-peptideagents that may be present at a ratio of at least 100:1, on aweight:weight basis, relative to the plurality of first peptide-taggedagents. In certain further embodiments, the plurality of firstanti-peptide agents may be positioned in the first divergent flow pathdownstream of the first release zone. In certain further embodiments,the plurality of first anti-peptide agents may be configured toimmobilize at least one of the plurality of first peptide-tagged agents.In certain embodiments, the divergent flow device may further comprise aplurality of second anti-peptide agents. In certain further embodiments,the plurality of second anti-peptide agents may be positioned in thesecond divergent flow path downstream of the second release zone. Incertain further embodiments, the plurality of second anti-peptide agentsmay be configured to immobilize at least one of the plurality of secondpeptide-tagged agents.

In certain embodiments, the plurality of first anti-peptide agents maybe positioned in a first detection zone. In certain embodiments, theplurality of second anti-peptide agents may be positioned in a seconddetection zone. In certain further embodiments, the first detection zoneand the second detection zone may be in fluid communication. In certainfurther embodiments, the first detection zone and the second detectionzone may not be in fluid communication. In certain further embodiments,the first detection zone and the second detection zone may be indifferent sections of the divergent flow device. In certain embodiments,the first divergent flow path and the second divergent flow path may bein different sections of the divergent flow device.

Certain embodiments may provide, for example, a divergent flow device,comprising: (i) a central sample zone configured to release a samplecontaining a plurality of analytes into a first divergent flow path anda second divergent flow path; (ii) a first release zone in the firstdivergent flow path configured to release a plurality of firstpeptide-tagged agents into the first divergent flow path; (iii) a secondrelease zone in the second divergent flow path configured to release aplurality of second peptide-tagged agents into the second divergent flowpath; (iv) a plurality of first anti-peptide agents present at a ratioof at least 100:1, on a weight:weight basis, relative to the pluralityof first peptide-tagged agents, said plurality of first anti-peptideagents: (a) positioned in the first divergent flow path downstream ofthe first release zone; and (b) capable of immobilizing at least one ofthe plurality of first peptide-tagged agents; and (v) a plurality ofsecond anti-peptide agents positioned in the second divergent flow pathdownstream of the second release zone and the second release zone, saidplurality of second anti-peptide agents capable of immobilizing at leastone of the plurality of second peptide-tagged agents.

Certain embodiments may provide a method of making a lateral flowdevice. In certain embodiments, the method of making may comprisestriping a plurality of peptide-tagged agents on a first portion of apermeable assembly. In certain embodiments, the method of making maycomprise binding a plurality of anti-peptide agents to a second portionof the permeable assembly. In certain embodiments, the plurality ofanti-peptide agents may be present on the test strip at a ratio of atleast 100:1, on a weight:weight basis, relative to the plurality ofpeptide-tagged agents. In certain embodiments, the second portion of thepermeable assembly may be in fluid communication with the first portionof the permeable assembly.

Certain embodiments may provide, for example, a method of making alateral flow device, comprising: (i) binding a plurality of anti-peptideagents to a portion of a permeable assembly; (ii) striping a pluralityof peptide-tagged agents on a second portion of the permeable assembly,said second portion of the permeable assembly in fluid communicationwith the first portion of the permeable assembly, at least one of saidplurality of peptide-tagged agents forming a specific binding pair withat least one of said plurality of anti-peptide agents, wherein saidplurality of anti-peptide agents are present on the test strip at aratio of at least 100:1, on a weight:weight basis, relative to thepeptide-tagged agent.

Certain embodiments may provide a method of making a plurality oflateral flow devices. In certain embodiments, the method may comprisepreparing a quantity of universal detection members. In certainembodiments, the method may further comprise preparing a quantity ofrelease members. In certain embodiments, the method may furthercomprise, for example, assembling into fluid communication each of thequantity of release members with a different one of the quantity ofuniversal detection members. In certain embodiments, each of theuniversal detection members may have a common type of anti-peptideimmobilization agent. In certain embodiments, preparing the quantity ofuniversal detection members may comprise, for example, immobilizing adifferent plurality of the common type of anti-peptide agent onto one ormore sheets, for example one sheet, and partitioning the one or moresheets to form the plurality of universal detection members. In certainembodiments, preparing the quantity of release members may comprise, forexample, striping a plurality of a different type of peptide-taggedagent onto one or more sheets, for example one seet, and partitioningthe one or more sheets to form the plurality of release members. Incertain further embodiments, each of the different type ofpeptide-tagged agent may form a specific binding pair with the commontype of anti-peptide agent. In certain embodiments, each of thedifferent plurality of the common type of anti-peptide agent may bepresent at a ratio of at least 100:1, on a weight:weight basis, relativeto each of the plurality of the different type of peptide-tagged agent.In certain embodiments, at least one of the different plurality of thecommon type of anti-peptide agent may be present at a ratio of at least100:1, on a weight:weight basis, relative to at least one of theplurality of the different type of peptide-tagged agent.

In certain embodiments, each one of the plurality of a different type ofpeptide-tagged agent may comprise a different plurality of peptide tags.In certain further embodiments, at least one of each of the differentplurality of peptide tags may be a type of peptide tag which may be abinding partner with the common type of anti-peptide agent.

In certain embodiments, an apparatus, lateral flow device, assembly,permeable assembly, divergent flow device, lateral flow test kit, ormulti-purpose lateral flow test kit may comprise a control zone. Incertain further embodiments, the control zone may be configured toprovide an indication that the apparatus has functioned. In certainfurther embodiments, the control zone may be configured tonon-selectively capture species present in the sample, for exampleproteins, antibodies, macromolecules, particles, and the like. Incertain embodiments, the control zone may comprise, for example, proteinA and/or protein G.

Certain embodiments may provide, for example, a method of making aplurality of lateral flow devices, comprising: (i) preparing a quantityof universal detection members having a common type of anti-peptideimmobilization agent, comprising: immobilizing a different plurality ofthe common type of anti-peptide agent to each of the plurality ofuniversal detection members; (ii) preparing a quantity of releasemembers, comprising: striping a plurality of a different type ofpeptide-tagged agent on each of the plurality of release members, eachof the different type of peptide tagged agent forming a specific bindingpair with the common type of anti-peptide agent; and (iii) assemblinginto fluid communication each of the quantity of release members with adifferent one of the quantity of universal detection members, wherein atleast one of the different plurality of the common type of anti-peptideagent is present at a ratio of at least 100:1, on a weight:weight basis,relative to at least one of the plurality of the different type ofpeptide-tagged agent.

Certain embodiments may provide, for example, a lateral flow device,comprising: (i) a flow path defined by a permeable sub-assembly of thelateral flow device; (ii) a release zone comprising: (a) a plurality offirst peptide-tagged agents, said release zone configured to release atleast a portion of said plurality of first peptide-tagged agents intothe flow path; and (b) a plurality of second peptide-tagged agents, saidrelease zone configured to release at least a portion of said pluralityof second peptide-tagged agents into the flow path; and (iii) adetection zone comprising: (a) a plurality of first anti-peptide agentspresent in the flow path at a ratio of at least 100:1, on aweight:weight basis, relative to the plurality of first peptide-taggedagents, wherein at least one of said plurality of first anti-peptideagents is a binding partner with at least one of the configured to bereleased at least a portion of said plurality of first peptide-taggedagents; and (b) a plurality of second anti-peptide agents present in theflow path at a ratio of at least 100:1, on a weight:weight basis,relative to the plurality of second peptide-tagged agents, wherein atleast one of said plurality of second anti-peptide agents is a bindingpartner with at least one of the configured to be released at least aportion of said plurality of second peptide-tagged agents.

Certain embodiments may provide, for example, an assay platform fordetecting a plurality of analytes, comprising: (i) a permeable releasezone configured to transport a sample comprising a first analyte and asecond analyte, said permeable zone comprising: (a) a plurality of firstpeptide-tagged agents, at least a portion of said plurality of firstpeptide-tagged agents capable of forming a first analyte complexcomprising the first analyte; and (b) a plurality of secondpeptide-tagged agents, at least a portion of said plurality of secondpeptide-tagged agents capable of forming a second analyte complexcomprising the second analyte; and (ii) a permeable detection zoneconfigured to receive at least a portion of transported sample, thepermeable detection zone comprising: (a) a plurality of firstanti-peptide agents present at a ratio of at least 100:1, on aweight:weight basis, relative to the plurality of first peptide-taggedagents, said plurality of first anti-peptide agents configured toimmobilize at least one of the at least a portion of said plurality offirst peptide-tagged agents; and (b) a plurality of second anti-peptideagents configured to immobilize at least one of the at least a portionof said plurality of second peptide-tagged agents.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic view of lateral flow detection apparatus.

FIG. 2: Schematic view of solvent-driven lateral flow detectionapparatus.

FIG. 3: Schematic view of multi-purpose lateral flow test kit.

FIG. 4: Schematic view of radial flow detection apparatus.

FIG. 5: Schematic view of multi-analyte lateral flow detectionapparatus.

DETAILED DESCRIPTION OF THE INVENTION

Certain embodiments provide lateral flow apparatuses and systems todetect one or more analytes, for example: assay platforms, lateral flowdevices, assemblies, single-purpose kits, multi-purpose kits, anddivergent flow devices. Other types of lateral flow apparatuses arecontemplated herein. Certain embodiments provide lateral flow methods todetect one or more analytes, including, for example, methods ofdetection, methods of making lateral flow apparatuses, and methods ofmaking lateral flow kits. Other types of lateral flow methods arecontemplated herein.

Suitable analytes may include any analyte to which a peptide-taggedagent is able to bind (for example the peptide-tagged agent may bedesigned or selected to bind a pre-determined analyte). In certainembodiments, the analyte may be a predetermined analyte, for example ananalyte for which a specific type of peptide-tagged agent is selected.In certain embodiments, the analyte may comprise, for example, aprotein, microbe or a fragment thereof, a virus or a fragment thereof, apeptide, a biomarker, an antibody (for example, an antibody to aninfectious agent) or a fragment thereof, a nucleic acid, amacromolecule, a small molecule, a drug, a hormone (for example, humanchorionic gonadotrophin) or a fragment thereof, a hapten or a fragmentthereof, etc. In certain embodiments, the analyte may be any analytewhich has heretofore been assayed using known immunoassay procedures, orknown to be detectable by such procedures (see, for example, “TheImmunoassay Handbook,” Fourth Ed., D. Wild ed., Elsevier Ltd. (2013)).Other types of analytes are also contemplated herein.

Protein analytes may include, for example, a phosphoprotein. A range ofphosphoproteins are known including, for example, phosphorylated ERK, S6p240/44, AKT pT308 or AKT pS473. In certain embodiments, the analyte maycomprise, for example, a component of a cell signaling pathway, acytokine, or a tumour suppressor. In certain embodiments, the analytemay comprise, for example, a protein that is phosphorylated, methylated,or glycosylated, etc. In certain embodiments, the analyte may comprise,for example, phospho-ERK 1/2; total ERK 1/2; phospho-Akt 1/2/3; totalAkt 1/2/3; phospho-NF- Kr3 p65; total NF-K13 p65; phospho-l-kB;total-kBP; phospho-STAT3; total STAT3; phospho-STAT5 A/B; phospho-JNK1/2/3; total JNK 1/2/3; phospho-p38 MAPK; total p38 MAPK; phospho-p53;total p53; phospho-p70S6K; total p70S6K; and GAPDH. In otherembodiments, the protein may comprise, for example, an acute phaseprotein, for example a C-reactive protein, haptoglobin, hemopexin,alpha-1 acid glycoprotein, clusterin, alpha-2-macroglobulin, serumamyloid A (SAA) or serum amyloid P (SAP) in species including but notlimited to human, mouse, rat, rabbit, cat, dog, pig, cow, chicken, andmonkey. In certain embodiments, the protein may comprise, for example, aparticular form or state of a protein, an endogenous protein, or atransfected protein.

In certain embodiments, the analyte may comprise, for example, abiomarker, for example a cancer biomarker (for example a prostate cancerbiomarker or a bowel cancer biomarker) or a diabetic kidney diseasebiomarker. In certain embodiments, the analyte may comprise, forexample, an analyte useful for detection in plant or contaminantdiagnostics. In certain other embodiments, the analyte may comprise, forexample, an analyte useful for detection in human or veterinarydiagnostics. In certain embodiments, the analyte may be capable ofmigrating, dissolving, disconnecting, or diffusing from the sample intoa solvent. Suitable solvents may comprise, for example, water, analcohol, a buffered solution, a pH-balanced solution, an organicsolvent, or a combination of two or more thereof.

In certain embodiments, the concentration of the analyte or any one of aplurality of analytes may be in a range of 0-100 ng/mL, for example,between 1-100 ng/mL, between 1-50 ng/mL, between 1-25 ng/mL, between10-50 ng/mL, of in a range of 1-10 ng/mL. In certain embodiments, theconcentration of the analyte, the one or more analytes may be below 1ng/mL. In certain embodiments, the concentration of the analyte or anyone of a plurality of analytes may be above 50 ng/mL. In certainembodiments, the concentration of the analyte or any one of a pluralityof analytes may be 100 ng/mL or less, for example 50 ng/mL or less, 10ng/mL or less, 1 ng/mL or less, 100 pg/mL or less, or 10 pg/mL or less,1 pg/mL or less, 100 fg/mL or less, 10 fg/mL or less, or 1 fg/mL orless. In certain embodiments, the concentration of the analyte or anyone of a plurality of analytes may be greater than 10 ng/mL, greaterthan 1 ng/mL, greater than 100 pg/mL or, greater than 10 pg/mL, greaterthan 1 pg/mL, greater than 100 fg/mL, greater than 10 fg/mL, or greaterthan 1 fg/mL. In certain embodiments, the concentration of the analyteor any one of a plurality of analytes may be in the range of 1 fg/mL to100 ng/mL, for example 1 fg/mL to 10 ng/mL, 1 fg/mL to 1 ng/mL, 10 fg/mLto 100 ng/mL, 10 fg/mL to 10 ng/mL, 10 fg/mL to 1 ng/mL, 100 fg/mL to100 ng/mL, 100 fg/mL to 10 ng/mL, 100 fg/mL to 1 ng/mL, 1 pg/mL to 100ng/mL, 1 pg/mL to 10 ng/mL, or in the range of 1 pg/mL to 1 ng/mL.

In certain embodiments, the concentration of the analyte or any one of aplurality of analytes may be in a range of 0 mIU/mL (milli-InternationalUnits per milli-liter) to 100 mIU/mL, for example 0.25 mIU/mL to 0.5mIU/mL, 0.5 mIU/mL to 1 mIU/mL, 1 mIU/mL to 2 mIU/mL, 2 mIU/mL to 5mIU/mL, 5 mIU/mL to 10 mIU/mL, 10 mIU/mL to 15 mIU/mL, 15 mIU/mL to 25mIU/mL, 25 mIU/mL to 50 mIU/mL, or in a range of 50 mIU/mL to 100mIU/mL. In certain embodiments, the concentration of the analyte or anyone of a plurality of analytes may be less than 100 mIU/mL, less than 50mIU/mL, less than 25 mIU/mL, less than 15 mIU/mL, less than 10 mIU/mL,less than 5 mIU/mL, less than 2 mIU/mL, less than 1 mIU/mL, less than0.5 mIU/mL, or less than 0.25 mIU/mL.

In certain embodiments, the analyte may have a molecular weight in arange of 10 Da to 10,000,000 kDa, for example a molecular weight in arange of 10 Da to 25 Da, 25 Da to 75 Da, 75 Da to 100 Da, 100 Da to 200Da, 200 Da to 300 Da, 300 Da to 500 Da, 500 Da to 750 Da, 750 Da to 200Da, 100 Da to 1 kDa, 1 kDa to 10 kDa, 10 kDa to 15 kDa, 15 kDa to 20kDa, 20 kDa to 25 kDa, 25 kDa to 50 kDa, 50 kDa to 75 kDa, 75 kDa to 100kDa, 100 kDa to 125 kDa, 125 kDa to 150 kDa, 150 kDa to 175 kDa, 175 kDato 200 kDa, 200 kDa to 250 kDa, 250 kDa to 500 kDa, 500 kDa to 1,000kDa, 1,000 kDa to 5,000 kDa, 5,000 kDa to 10,000 kDa, 10,000 KDa to1,000,000 kDa, 1,000,000 kDa to 10,000,000 kDa, 75 kDa to 400 kDa, 100kDa to 200 kDa, or a molecular weight in a range of 125 kDa to 175 kDa.

Certain embodiments may provide an assay platform for detecting at leastone type of an analyte in a sample, optionally in the presence of one ormore type of non-analyte antibody. In certain embodiments, for example,the assay platform may comprise, for example, at least one type ofpeptide-tagged agent and at least one type of anti-peptide tag. Incertain embodiments, the affinity, or rate constant, for binding one ofthe at least one type of anti-peptide agent to one of the at least onetype of peptide-tagged agent may be approximately the same as theaffinity, or rate constant, for binding the one of the at least one typeof anti-peptide agent to any of the one or more type of non-analyteantibody. In certain embodiments, the affinity, or rate constant, forbinding one of the at least one type of anti-peptide agent to one of theat least one type of peptide-tagged agent may be at least 2 times largerthan the affinity, or rate constant, for binding the one of the at leastone type of anti-peptide agent to any of the one or more type ofnon-analyte antibody, for example at least 5, 10, 25, 50, 75, or atleast 100 times larger. In certain embodiments, the affinity, or rateconstant, for binding one of the at least one type of anti-peptide agentto one of the at least one type of peptide-tagged agent may be in therange of 2-1000 times larger than the affinity, or rate constant, forbinding the one of the at least one type of anti-peptide agent to any ofthe one or more type of non-analyte antibody, for example in the rangeof 2-500, 2-100, 2-10, 10-250, 20-100, or in the range of 5-50 timeslarger.

In certain embodiments, the dissociation constant between one of the atleast one type of anti-peptide agent and one of the at least one type ofpeptide-tagged agent may be approximately the same as the dissociationconstant between one of the at least one type of anti-peptide agent andany of the one or more type of non-analyte antibody. In certainembodiments, the dissociation constant between one of the at least onetype of anti-peptide agent and one of the at least one type ofpeptide-tagged agent may be at least 2 times larger than thedissociation constant between one of the at least one type ofanti-peptide agent and any of the one or more type of non-analyteantibody, for example at least 5, 10, 25, 50, 75, or at least 100 timeslarger. In certain embodiments, the dissociation constant between one ofthe at least one type of anti-peptide agent and one of the at least onetype of peptide-tagged agent may be in the range of 2-1000 times largerthan the dissociation constant between one of the at least one type ofanti-peptide agent and any of the one or more type of non-analyteantibody, for example in the range of 2-500, 2-100, 2-10, 10-250,20-100, or in the range of 5-50 times larger.

In certain embodiments, one of the at least one type of peptide-taggedagent may form a first specific binding pair with one of one or morepre-determined type of analyte, wherein said specific binding pair mayhave a dissociation constant (Kd) of greater than 10⁻⁶M. In furtherembodiments, said first specific binding pair may have a Kd of greaterthan 10⁻⁷M, 10⁻⁸M or 10⁻⁹M. In certain embodiments, said first specificbinding pair may have a dissociation constant (Kd) in the range from10⁻⁸M to 10⁻¹²M. In certain embodiments, the one of the at least onetype of peptide-tagged agent may form a second specific binding pairwith one of the at least one type of anti-peptide agent, wherein saidsecond specific binding pair may have a dissociation constant (Kd) ofgreater than 10⁻⁶M. In further embodiments, said second second specificbinding pair may have a Kd of greater than 10⁻⁷M, 10⁻⁸M or 10⁻⁹M. Incertain embodiments, said second specific binding pair may have adissociation constant (Kd) in the range from 10⁻⁸M to 10⁻¹²M. In certainembodiments, the first specific binding pair may have a Kd in the rangeof 10⁻⁸M to 10⁻¹²M and the second specific binding pair may have a Kd inthe range of greater than 10⁻⁶M. In certain embodiments, the secondspecific binding pair may have a Kd in the range of 10⁻⁸M to 10⁻¹²M andthe first specific binding pair may have a Kd in the range of greaterthan 10⁻⁶M.

In certain embodiments, one of the at least one type of peptide-taggedagent may be present in the assay platform in an amount in the range ofbetween 1 ng and 50 ng, for example in the range of between 3 ng and 30ng, 4 ng and 20 ng, 4 ng and 15 ng, 5 ng and 10 ng, or in the range ofbetween 5 ng and 8 ng, for example 6.5 ng. In certain embodiments, oneof the least one type of peptide-tagged agent may be present in theassay platform in an amount of less than 10 ng, for example less than 9ng, 8 ng, 7.5 ng, 7 ng, 6.5 ng, 6 ng, 5.5 ng, 5 ng, 4.5 ng, 4 ng, 3.5ng, 3 ng, 2.5 ng, or in an amout of less than 2 ng.

In certain embodiments, one of the at least one type of peptide-taggedagent may be present in the assay platform in an amount in the range ofbetween 10 ng/cm² and 500 ng/cm² of test strip area striped by the oneof the at least one type of peptide-tagged agent, for example in therange of between 30 ng/cm² and 300 ng/cm², 40 ng/cm² and 200 ng/cm², 40ng/cm² and 150 ng/cm², 50 ng/cm² and 100 ng/cm², or in the range ofbetween 50 ng/cm² and 80 ng/cm², for example 65 ng/cm² of test striparea striped by the one of the at least one type of peptide-taggedagent. In certain embodiments, one of the at least one type ofpeptide-tagged agent may be present in the assay platform in an amountof less than 100 ng/cm² of test strip area striped by the one of the atleast one type of peptide-tagged agent, for example less than 90 ng/cm²,80 ng/cm², 75 ng/cm², 70 ng/cm², 65 ng/cm², 60 ng/cm², 55 ng/cm², 50ng/cm², 45 ng/cm², 40 ng/cm², 35 ng/cm², 30 ng/cm², 25 ng/cm², or in anamout of less than 20 ng/cm² of test strip area striped by the one ofthe at least one type of peptide-tagged agent. In certain furtherembodiments, the test strip area striped by the one of the at least onetype of peptide-tagged agent may have a surface area in the range of0.01- 5 cm², for example 0.1-2 cm², 0.15-2 cm², or a surface area in therange of 0.25-1 cm².

Certain embodiments may provide a lateral flow apparatus or system todetect an analyte. The lateral flow apparatus or system may comprise,for example, a plurality of peptide-tagged agents. In certainembodiments, the plurality of peptide-tagged agents may be capable offorming an analyte complex that may comprise at least one of theplurality of peptide-tagged agents and the analyte. In certainembodiments, the lateral flow apparatus or system may further comprise,for example, a plurality of anti-peptide agents present, for example, ata ratio of at least 100:1, on a weight:weight basis, relative to theplurality of peptide-tagged agents. In certain embodiments, the lateralflow apparatus or system may further comprise, for example, a pluralityof detectable agents, and, optionally, the analyte complex may furthercomprise, for example, at least one of the plurality of detectableagents.

In certain embodiments, the sample may be any mixture, composition, orsolution that may or may not contain the analyte. In certain assays, itmay be useful, for example, to determine that an analyte may not bepresent in a sample. In certain embodiments, the sample may comprise,for example, a laboratory sample, a medical sample, a biological sample,a water sample, a food sample, an agricultural sample, a serum sample, aserum containing sample, a cell lysate, or a combination of two or morethereof. The sample may comprise, for example, urine, blood, or anotherbodily fluid of a human or animal.

In certain embodiments, the sample may be a liquid, for example anaqueous solution, a suspension, or a liquid mixture; a solid; a gel; orcongealed matter. In certain embodiments, the sample may be dissolved,mixed, passively mixed, or diffused in a solvent. In certainembodiments, the sample may be pretreated, for example the sample may beprecleared, concentrated, diluted, or processed to remove one or morecomponents or impurities from the sample.

A peptide-tagged agent may comprise, for example, an agent that may becapable of binding with the analyte, for example an antibody, inclusive,for example, of a monoclonal antibody, a polyclonal antibody, amultivalent antibody, a chimeric antibody, a multispecific antibody, oran antibody fragment; an aptamer; an affimer; a protein; a proteinreceptor; a protein ligand; or a fusion protein, comprising, forexample, an immunoglobulin fusion partner, a fusion partner thatstabilizes a receptor or a ligand, or a fusion partner that provides atarget for binding.

In certain embodiments, the peptide-tagged agent may have a molecularweight in a range of 100 Da to 10,000 kDa, for example a molecularweight in a range of 100 Da to 1 kDa, 1 kDa to 10 kDa, 10 kDa to 25 kDa,25 kDa to 50 kDa, 50 kDa to 75 kDa, 75 kDa to 100 kDa, 100 kDa to 125kDa, 125 kDa to 150 kDa, 150 kDa to 175 kDa, 175 kDa to 200 kDa, 200 kDato 250 kDa, 250 kDa to 500 kDa, 500 kDa to 1,000 kDa, 1,000 kDa to 5,000kDa, 5,000 kDa to 10,000 kDa, 75 kDa to 400 kDa, 100 kDa to 200 kDa, ora molecular weight in a range of 125 kDa to 175 kDa.

In certain embodiments, the peptide-tagged agent may be capable ofbinding to an epitope of the analyte, for example the peptide-taggedagent may comprise a complementary determinant region, or a portionthereof, that may be capable of binding with one or more epitopes of theanalyte, for example a phospho-epitope. In certain embodiments, theanalyte may be a target for the peptide-tagged agent. In certainembodiments, the peptide-tagged agent may be non-covalently bound to theanalyte, for example releasably bound, partially bonded,hydrogen-bonded, ionically bonded, or a combination of two or morethereof.

In certain embodiments, the peptide-tagged agent may further comprise,for example, a plurality of peptide tags. In certain embodiments, thepeptide-tagged agent may further comprise, for example, a plurality of asingle type of peptide tag. In certain embodiments, the peptide-taggedagent may further comprise, for example, a plurality of two, three, orfour types of peptide tags. In certain embodiments, the plurality ofpeptide tags may be attached, conjugated, and/or covalently bound to thepeptide-tagged agent. In certain embodiments, the plurality of peptidetags may be bound to the peptide-tagged agent via cross-linking to thepeptide-tagged agent. In certain further embodiments, the cross-linkingmay comprise, for example, covalent conjugation by primary amines. Incertain embodiments, Recombinant DNA technology may be used to form apeptide-tagged agent comprising the plurality of peptide tags.

In certain embodiments, the plurality of peptide tags may comprise, forexample, in a range of 1-50 or 2-50 peptide tags, such as 5-8, 3-7, 2-5,10-15, 15-20, or in the range of 25-50 peptide tags. In certainembodiments, the plurality of peptide tags may comprise, for example, nomore than 50 peptide tags, such as no more than 12, no more than 10, nomore than 8, no more than 7, no more than 6, no more than 5, or no morethan 3 peptide tags. In certain embodiments, the plurality of peptidetags may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 peptide tags.

Suitable peptide tags may include a FLAG-tag, for example DYKDDDDK (SEQID NO: 1); a peptide tag having the amino acid sequenceKRITVEEALAHPYLEQYYDPTDE (SEQ ID NO: 2); a peptide tag having the aminoacid sequence HHHHHH (SEQ ID NO: 3); a peptide tag having the amino acidsequence EQKLISEEDL (SEQ ID NO: 4); a peptide tag having the amino acidsequence YPYDVPDYA (SEQ ID NO: 5); a peptide tag having the amino acidsequence YTDIEMNRLGK (SEQ ID NO: 6); a peptide tag having the amino acidsequence QPELAPEDPED (SEQ ID NO: 7); a peptide tag having the amino acidsequence CDYKDDDDK (SEQ ID NO: 8) a FLAG octapeptide; a polypeptideprotein tag; or a polypeptide sequence that does not include a pluralityof consecutive amino acids with the same charge. In certain furtherembodiments, the suitable peptide tags may also be used in conjunctionwith other affinity tags, for example a polyhistidine tag (His-tag),HA-tag or myc-tag. In certain embodiments, at least a portion of thepeptide tags may not be naturally occurring. In certain embodiments, thesuitable peptide tags do not denature or inactivate the peptide-taggedagent to which they are attached. In certain embodiments, at least oneof the suitable peptide tags is more hydrophilic than the FLAG-tag. Incertain embodiments, at least a portion of the suitable peptide tags maybe removed from the peptide-tagged agent to which they are attached bytreatment with the specific proteinase, enterokinase (enteropeptidase).In certain embodiments, at least a portion of the suitable peptide tagsmay not be naturally occurring in an organism from which a sample istaken. In certain embodiments, suitable peptide tags may include aminoacid sequences having no more than 100 amino acids, for example, no morethan 50, no more than 30, no more than 20, no more than 15, no more than12, no more than 10, no more than 9, or no more than 8 amino acids. Incertain embodiments, suitable peptide tags may include amino acidsequences having in the range of 4-100 amino acids, for example in therange of 4-50, 4-30, 4-20, 6-15, 6-12, 8-20, 8-15, or in the range of8-12 amino acids.

In certain embodiments, an anti-peptide agent may comprise, for example,an agent capable of binding with at least one of the plurality ofpeptide tags, for example at least 10%, 25%, 50%, 90%, or 95% of theplurality of peptide tags, such as for example, each of the plurality ofpeptide tags. In certain embodiments, a suitable anti-peptide agent maycomprise, for example, an antibody, inclusive, for example, of amonoclonal antibody, a polyclonal antibody, a multivalent antibody, achimeric antibody, a multispecific antibody, or an antibody fragment; anaptamer; an affimer; a protein; a protein receptor; a protein ligand; ora fusion protein, comprising, for example, an immunoglobulin fusionpartner, a fusion partner that stabilizes a receptor or a ligand, or afusion partner that provides a target for binding.

In certain embodiments, the anti-peptide agent may have a molecularweight in a range of 100 Da to 10,000 kDa, for example a molecularweight in a range of 100 Da to 1 kDa, 1 kDa to 10 kDa, 10 kDa to 25 kDa,25 kDa to 50 kDa, 50 kDa to 75 kDa, 75 kDa to 100 kDa, 100 kDa to 125kDa, 125 kDa to 150 kDa, 150 kDa to 175 kDa, 175 kDa to 200 kDa, 200 kDato 250 kDa, 250 kDa to 500 kDa, 500 kDa to 1,000 kDa, 1,000 kDa to 5,000kDa, 5,000 kDa to 10,000 kDa, 75 kDa to 400 kDa, 100 kDa to 200 kDa, ora molecular weight in a range of 125 kDa to 175 kDa.

In certain embodiments, at least one of the plurality of peptide tagsmay be a target for the anti-peptide agent. In certain embodiments, atleast one of the plurality of peptide tags and the anti-peptide agentmay be specific binding partners. In certain embodiments, theanti-peptide agent may comprise, for example, a binding region highlyselective for at least one of the peptide tags, or a portion of at leastone of the peptide tags, such as, for example for a peptide tag havingthe amino acid sequence DYKDDDDK (SEQ ID NO: 1); a peptide tag havingthe amino acid sequence CDYKDDDDK (SEQ ID NO: 8); a peptide tag havingthe amino acid sequence KRITVEEALAHPYLEQYYDPTDE (SEQ ID NO: 2; a peptidetag having the amino acid sequence HHHHHH (SEQ ID NO: 3); a peptide taghaving the amino acid sequence EQKLISEEDL (SEQ ID NO: 4); a peptide taghaving the amino acid sequence YPYDVPDYA (SEQ ID NO: 5); YTDIEMNRLGK(SEQ ID NO: 6); or a peptide tag having the amino acid sequenceQPELAPEDPED (SEQ ID NO: 7). For example, the binding region of theanti-peptide agent may be highly selective for at least 60% of aminoacid sequence of at least one of the peptide tags, such as 70%, 80%,90%, 95%, or 100%, of the amino acid sequence of at least one of thepeptide tags. Commercially available anti-peptide antibodies antibodiesthat are highly selective for a peptide tag having the amino acidsequence DYKDDDDK (SEQ ID NO: 1) include Sigma-Aldrich product codesF7425, F3040, F1804, F3165, F4042, F2555 and SAB4200071. In certainembodiments, the anti-peptide agent may be selective to a peptide tagonly when the peptide tag occupies a certain type of position on apeptide-tagged agent, for example a peptide attached at the N-terminalposition. In other embodiments, the anti-peptide agent may beinsensitive to how a peptide tag is positioned on an anti-peptide agent.

In certain embodiments, a plurality of anti-peptide agents may bepresent at a ratio of at least 100:1, 125:1, 150:1, 200:1, 250:1, 300:1,400:1, 500:1, 750:1, or 1000:1, on a weight:weight basis, relative tothe plurality of peptide-tagged agents. In certain embodiments, aplurality of anti-peptide agents may be present at a ratio of in a rangeof 100:1-1000:1, 100:1-500:1, 100:1-300:1, 250:1-300:1, 300:1-1000:1,300:1-500:1, 300:1-400:1, 300:1-350:1, 325:1-375:1, 300:1-310:1,310:1-320:1, 320:1-330:1, 330:1-340:1, 340:1-350:1, or 350:1-360:1, on aweight:weight basis, relative to the plurality of peptide-tagged agents.

In certain embodiments, a plurality of anti-peptide agents may bepresent at a ratio of at least 100:1, 125:1, 150:1, 200:1, 250:1, 300:1,400:1, 500:1, 750:1, or 1000:1, on a mole:mole basis, relative to theplurality of peptide-tagged agents. In certain embodiments, a pluralityof anti-peptide agents may be present at a ratio of in a range of100:1-1000:1, 100:1-500:1, 100:1-300:1, 250:1-300:1, 300:1-1000:1,300:1-500:1, 300:1-400:1, 300:1-350:1, 325:1-375:1, 300:1-310:1,310:1-320:1, 320:1-330:1, 330:1-340:1, 340:1-350:1, or 350:1-360:1, on amole:mole basis, relative to the plurality of peptide-tagged agents.

A detectable agent may comprise, for example, an agent that may becapable of binding with the analyte, for example an antibody, inclusive,for example, of a monoclonal antibody, a polyclonal antibody, amultivalent antibody, a chimeric antibody, a multispecific antibody, oran antibody fragment; an aptamer; an affimer; a protein; a proteinreceptor; a protein ligand; or a fusion protein, comprising, forexample, an immunoglobulin fusion partner, a fusion partner thatstabilizes a receptor or a ligand, or a fusion partner that provides atarget for binding, or a fusion partner that provides a detectablesignal. In certain embodiments, the detectable agent may be capable ofbinding to an epitope of the analyte, for example the detectable agentmay comprise a complementary determinant region, or a portion thereof,that may be capable of binding with one or more epitopes of the analyte,for example a phospho-epitope.

In certain embodiments, the detectable agent may have a molecular weightin a range of 100 Da to 10,000 kDa, for example a molecular weight in arange of 100 Da to 1 kDa, 1 kDa to 10 kDa, 10 kDa to 25 kDa, 25 kDa to50 kDa, 50 kDa to 75 kDa, 75 kDa to 100 kDa, 100 kDa to 125 kDa, 125 kDato 150 kDa, 150 kDa to 175 kDa, 175 kDa to 200 kDa, 200 kDa to 250 kDa,250 kDa to 500 kDa, 500 kDa to 1,000 kDa, 1,000 kDa to 5,000 kDa, 5,000kDa to 10,000 kDa, 75 kDa to 400 kDa, 100 kDa to 200 kDa, or a molecularweight in a range of 125 kDa to 175 kDa.

In certain embodiments, the analyte may be a target for the detectableagent. In certain embodiments, the detectable agent may benon-covalently bound to the analyte, for example releasably bound,partially bonded, hydrogen-bonded, ionically bonded, or a combination oftwo or more thereof.

In certain embodiments, one or more of the detectable agents maycomprise, for example, a detectable tag that may be capable of producinga detectable signal. In certain embodiments, the magnitude, or anothercharacteristic, of the signal may be related to the quantity orconcentration of an analyte in a sample. In certain embodiments, thedetectable tag may be applied to or bound to another portion of thedetectable agent to form the complete detectable agent. In certainembodiments, the detectable tag may be integral to the rest of thedetectable agent. For example, the detectable agent may include thedetectable tag as a fusion partner, a labelled amino acid, or a labellednucleotide.

Suitable detectable tags may include, for example, antigens; enzymes;fluorophores; quenchers; radioactive isotopes; luminescent labels;chemiluminescent labels; one or more lanthanide ions, for example Eu³⁺,Sm³⁺, Tb³⁺, and/or Dy³⁺, nucleic acids that may be capable of PCRamplification; coloured particles, inclusive, for example, of colouredlatex beads, metal sols (for example, collidal gold), gold labels, ordye labels; tags that may be capable of interaction by fluorescenceresonance energy transfer; and tags that may be capable of chemicaltransfer proximity interaction.

In certain embodiments, the detectable signal may be produced directlyby the detectable agent or detectable tag, if present, or indirectly bya further molecule that can produce a detectable signal, such as, forexample, a further molecule that may comprise an enzyme. In certainembodiments, detectable signals include, but are not limited to, lightsignals, inclusive of, for example, signals having a color or no colorthat may be detectable by human vision; stains; and signals that may bedetected with a detector, inclusive of, for example, aspectrophotometer, a fluorometer, for example a fluorometer capable ofmeasuring time-resolved fluorescence, a luminometer, aradioactivity-based counter, or an electrochemical detection apparatus.In certain embodiments, the detector may be a hand-held detector, adetector in a laboratory, and/or a mobile detector. In certainembodiments, detectable signals may include the lack of any of theforegoing types of signals, for example a quenched signal, when measuredby a detector capable of detecting a signal if one were present.

Metal sols and other types of colored particles useful as detectabletags in immunoassay procedures are known. See, for example, U.S. Pat.No. 4,313,734; Horisberger, Evaluation of Colloidal Gold as aCytochromic Marker for Transmission and Scanning Electron Microscopy,Biol. Cellulaire, 36, 253 258 (1979); Leuvering et al., Sol ParticleImmunoassay, J. Immunoassay, 1 (1): 77 91 (1980), and Frens, ControlledNucleation for the Regulation of the Particle Size in Monodisperse GoldSuspensions, Nature, Physical Science, 241: 20 22 (1973).

Use of metal sols, for example colloidal gold, may produce a visiblydetectable signal. In certain embodiments, detectable tags may comprise,for example, colloidal gold providing a red detectable signal. Incertain embodiments, detectable tags may comprise, for example,colloidal gold having a mean particle size in a range of 50-100 nm, suchas, for example, colloidal gold having a mean particle size in a rangeof 55-90, 55-85, 40-47, 60-80, 60-75, or 65-75 nm. In certainembodiments, detectable tags may comprise, for example, colloidal goldhaving a mean particle size greater than 50 nm, greater than 52 nm,greater than 55 nm, greater than 57 nm, greater than 60 nm, or greaterthan 100 nm in size.

In certain embodiments, detectable tags may comprise, for example,particles, at least a portion of which may be substantially orpredominantly spherical in shape. In certain embodiments, at least aportion of the detectable tags may include particles havingsubstantially or predominantly non-spherical shape or shapes. In certainembodiments, detectable tags may comprise, for example, particles thatmay be substantially or predominantly monodisperse or have a narrowparticle size distribution, for example a monodispersity greater than50%, 75% or 95% as determined by a Coulter N4 Particle Analyzer (BeckmanCoulter, Fullerton, Calif.).

Antigens that may be used as detectable tags may include, for example,any antigenic component of a detectable agent that may be targeted by asecondary deteclable agent. In certain embodiments, an antibody may beused as a secondary detectable agent to detect an antigen detectabletag. In certain further embodiments, the antibody secondary detectableagent may be fluorescently or enzymatically labelled. In embodimentswhere the portion of the detectable agent that may bind with the analyteis part of an antibody, the antibody secondary detectable agent may havea binding affinity to an antigen on the detectable agent.

Enzymes that may be used as detectable tags include, for example,enzymes that result in the conversion of a portion of the detectableagent into a detectable product. In certain embodiments, the conversionmay result in a change in colour or fluorescence or generation of anelectrochemical signal by the detectable agent. Such enzymes mayinclude, for example, horseradish peroxidase (HRP), alkaline phosphatase(AP), p-galactosidase, acetylcholinesterase, luciferase, or catalase.

Radioactive isotopes that may be used as detectable tags include, forexample, ³H, ¹⁴C_(,) ³² _(P,) ³⁵S, or ¹³¹I. In certain embodiments, theradioisotope may be conjugated to a detectable agent or incorporatedinto a detectable agent by translation of mRNA encoding the detectableagent in the presence of radiolabelled amino acids. Radioisotopes andmethods for conjugating radioactive isotopes to molecules such asproteins are known in the art and include methods discussed by Slater(Radioisotopes in Biology: A Practical Approach, Oxford UniversityPress, 2002). Radioisotopes may be detected using gamma, beta orscintillation counters.

Fluorophores that may be used as detectable tags include, for example,resorufin; fluorescein, inclusive of, for example, fluoresceinisothiocyanate, FITC; rhodamine, inclusive of, for example, tetramethylrhodamine isothiocyanate, TRITC; green fluorescent protein, GFP; andphycobiliproteins, inclusive of, for example, allophycocyanin,phycocyanin, phycoerythrin and phycoerythrocyanin, or derivatives of anyof the foregoing.

In certain embodiments, fluorophores may be subjected to appliedstimulation, for example light of a suitable excitation wavelength topromote fluorescence. Alternatively, in certain embodiments stimulationmay be provided by a fluorescence resonance energy transfer (FRET)partner, for example a donor molecule. When the fluorophore comes intoclose vicinity to the FRET partner, for example during formation of theanalyte complex, the fluorophore may become excited by the FRET partnerand fluoresce. FRET donors may include luminescent and/or fluorescentagents.

In certain embodiments, a detectable tag may comprise, for example, aquencher. Quenchers may be able to absorb excitation energy fromfluorophores and may be used to suppress the fluorophore's emission whenin close proximity. In this regard, the reaction is similar to a FRETreaction, except that the readout is a loss of fluorescence.

Luminescent compounds that may be used as detectable tags include, forexample, chemiluminescent and bioluminescent compounds. In certainembodiments, these compounds may be used to label the detectable agent.In certain embodiments, the presence of the chemiluminescent-tag may bedetermined by detecting the presence of luminescence that arises duringthe course of a chemical reaction. Examples of useful chemiluminescentlabelling compounds include, but are not limited to, luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester. In certain embodiments, the presence of a bioluminescentantibody is determined by detecting the presence of luminescence.Examples of bioluminescent compounds include, but are not limited toluciferin, luciferase and aequorin.

Nucleic acids that may be used as detectable tags include a suitablenucleic acid that may be capable of PCR amplification and/orhybridisation to a probe. The nucleic acid may be of sufficient lengthto allow binding of a forward and/or a reverse primer. In certainembodiments, the nucleic acid tag may be included in an aptamer or boundto a protein. In certain embodiments, nucleic acid detectable tags maybe detected by performing a PCR reaction, whereby the nucleic acid tagis amplified and measured, or using a labelled nucleic acid probe with acomplementary sequence to at least a portion of the nucleic acid tag.Methods of preparing and binding detectable nucleic acid tags todetectable agents are known in the art and include methods described inUS 2009/0053701.

Detectable tags that may be capable of detection derived from chemicaltransfer proximity interaction may comprise, for example, a photoactiveindicator precursor, for example a photoactive indicator precursor boundto a particle (such as a bead), which may be activated (for example,activated by singlet oxygen) to form a photoactive indicator. In certainembodiments, the photoactive indicator may be stimulated (for example,by irradiation) to produce a measurable light signal. Suitablephotoactive indicator precursors may include, for example, rubrene,europium, europium chelate, samarium, or terbium. In embodiments where aphotoactive indicator precursor is bound to a bead, suitable beads mayinclude, for example, latex beads and magnetic beads.

In certain embodiments, a photoactive indicator may be capable ofproducing a light signal when irradiated by light. In certain furtherembodiments, the irradiating light may have a wavelength in a range of250-1100 nm, for example, light at a wavelength in a range of 300-1000nm, between 450-950 nm, between 360-441 nm, between 620-700 nm, between600-630 nm, between 620-650 nm, between 640-700 nm, between 650-700 nm,between 670-690 nm, between 680-700 nm, or between 660-680 nm, such asby light at a wavelength of 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670nm, 675 nm, 680 nm, 685 nm, 690 nm, or at a wavelength of 700 nm.

In certain embodiments, the photoactive indicator, when irradiated, mayfluoresce (i.e., emit a fluorescence) at a wavelength in a range of500-625 nm, such as at a wavelength in a range of 525-575 nm, between525-550 nm, between 540-560 nm, between 540-550 nm, 590-620 nm, between600-625 nm, or in the range of 610-620 nm, such as at a wavelength of520 nm, 530 nm, 535 nm, 540 nm, 545 nm, 550 nm, 555 nm, 560 nm, 600 nm,605 nm, 610 nm, 615 nm, 620 nm, or at a wavelength of 625 nm.

In certain embodiments, detection of an analyte may comprise detecting aloss or gain of a light emission that may be derived from an interactionbetween at least a portion of the plurality of detectable tags and atleast a portion of the permeable detection members. In certainembodiments, for example in certain embodiments utilizing a quencher, aFRET fluorophore, or photoactive indicator precursor as at least one ofthe detectable tags, an interacting component may be provided on, orintegrated with, at least a portion of the permeable detection member.For example, in certain embodiments where at least a portion of thedetectable tags comprise a fluorophore, the at least a portion of thepermeable detection member may comprise a FRET partner (either donor oracceptor) or a quencher. In certain embodiments, in other embodimentswhere at least a portion of the plurality of detectable tags comprises aquencher, the at least a portion of the permeable detection member maycomprise a suitable fluorophore. In certain embodiments, at least aportion of the plurality of detectable tags may comprise a chemicaltransfer interaction acceptor, for example a photoactive indicatorprecursor and, optionally, at least a portion of the permeable detectionmembers may comprise a chemical transfer interaction donor, for examplea donor capable of providing singlet oxygen upon stimulation by anappropriate light source. In certain embodiments, at least a portion ofthe plurality of permeable detection members may comprise a chemicaltransfer interaction acceptor, for example a photoactive indicatorprecursor and, optionally, at least a portion of the detectable tags maycomprise a chemical transfer interaction donor, for example a donorcapable of providing singlet oxygen upon stimulation by an appropriatelight source.

In certain embodiments, detecting the presence of the immobilisedanalyte complex on at least a portion of the permeable detectionmembrane may utilise time-resolved fluorescence (TRF) and (fluorescenceresonance energy transfer) FRET technologies, inclusive of, for example,the TRF-FRET technologies as described in EP 569,496, U.S. Pat. No.5,527,684 or U.S. Pat. No. 6,861,264.

In certain embodiments, a plurality of anti-peptide agents may bepresent at a ratio of at least 0.1:1, 1:1, 2:1, 5:1, 8:1, 10:1, 15:1,25:1, 50:1, 100:1, 300:1, or 500:1, on a weight:weight basis, relativeto a plurality of detectable agents. In certain embodiments, a pluralityof anti-peptide agents may be present at a ratio of in a range of0.1:1-500:1, 0.1:1-300:1, 0.1:1-300:1, 0.1:1-500:1, 2:1-500:1,2:1-100:1, 2:1-50:1, 2:1-25:1, 2:1-15:1, 5:1-15:1, 5:1-15:1, 5:1-10:1,5:1-8:1, or 8:1-10:1, on a weight:weight basis, relative to theplurality of detectable agents.

In certain embodiments, a plurality of anti-peptide agents may bepresent at a ratio of at least 0.1:1, 1:1, 2:1, 5:1, 8:1, 10:1, 15:1,25:1, 50:1, 100:1, 300:1, or 500:1, on a mole:mole basis, relative to aplurality of detectable agents. In certain embodiments, a plurality ofanti-peptide agents may be present at a ratio of in a range of0.1:1-500:1, 0.1:1-300:1, 0.1:1-300:1, 0.1:1-500:1, 2:1-500:1,2:1-100:1, 2:1-50:1, 2:1-25:1, 2:1-15:1, 5:1-15:1, 5:1-15:1, 5:1-10:1,5:1-8:1, or 8:1-10:1, on a mole:mole basis, relative to the plurality ofdetectable agents.

In certain embodiments, a plurality of detectable agents may be presentat a ratio of at least 0.1:1, 1:1, 2:1, 5:1, 8:1, 10:1, 15:1, 25:1,50:1, 100:1, 300:1, or 500:1, on a weight:weight basis, relative to aplurality of peptide-tagged agents. In certain further embodiments, theplurality of detectable agents may be present at a ratio of in a rangeof 0.1:1-500:1, 0.1:1-300:1, 0.1:1-300:1, 0.1:1-500:1, 2:1-500:1,2:1-100:1, 2:1-50:1, 10:1-50:1, 20:1-50:1, 25:1-45:1, 30:1-45:1,30:1-40:1, 35:1-40:1, or 37:1-40:1, on a weight:weight basis, relativeto a plurality of peptide-tagged agents .

In certain embodiments, a plurality of detectable agents may be presentat a ratio of at least 0.1:1, 1:1, 2:1, 5:1, 8:1, 10:1, 15:1, 25:1,50:1, 100:1, 300:1, or 500:1, on a weight:weight basis, relative to aplurality of peptide-tagged agents. In certain further embodiments, theplurality of detectable agents may be present at a ratio of in a rangeof 0.1:1-500:1, 0.1:1-300:1, 0.1:1-300:1, 0.1:1-500:1, 2:1-500:1,2:1-100:1, 2:1-50:1, 10:1-50:1, 20:1-50:1, 25:1-45:1, 30:1-45:1,30:1-40:1, 35:1-40:1, or 37:1-40:1, on a weight:weight basis, relativeto a plurality of peptide-tagged agents.

In certain embodiments, a plurality of anti-peptide agents may bepresent at a ratio in a range of 5:1-20:1, on a weight:weight basis,relative to the plurality of detectable agents, and the plurality ofanti-peptide agents may be present at a ratio of in a range of100:1-500:1, on a weight:weight basis, relative to a plurality ofpeptide-tagged agents. In certain embodiments, a plurality ofanti-peptide agents may be present at a ratio in a range of 5:1-10:1, ona weight:weight basis, relative to the plurality of detectable agentsand the plurality of anti-peptide agents may be present at a ratio of ina range of 150:1-400:1, on a weight:weight basis, relative to aplurality of peptide-tagged agents. In certain embodiments, a pluralityof anti-peptide agents may be present at a ratio in a range of 5:1-10:1,on a weight:weight basis, relative to the plurality of detectable agentsand the plurality of anti-peptide agents may be present at a ratio of ina range of 200:1-500:1, on a weight:weight basis, relative to aplurality of peptide-tagged agents. In certain embodiments, a pluralityof anti-peptide agents may be present at a ratio in a range of 6:1-10:1,on a weight:weight basis, relative to the plurality of detectable agentsand the plurality of anti-peptide agents may be present at a ratio of ina range of 250:1-400:1, on a weight:weight basis, relative to aplurality of peptide-tagged agents.

In certain embodiments, a plurality of anti-peptide agents may bepresent at a ratio in a range of 5:1-20:1, on a mole:mole basis,relative to the plurality of detectable agents, and the plurality ofanti-peptide agents may be present at a ratio of in a range of100:1-500:1, on a mole:mole basis, relative to a plurality ofpeptide-tagged agents. In certain embodiments, a plurality ofanti-peptide agents may be present at a ratio in a range of 5:1-10:1, ona mole:mole basis, relative to the plurality of detectable agents, andthe plurality of anti-peptide agents may be present at a ratio of in arange of 150:1-400:1, on a mole:mole basis, relative to a plurality ofpeptide-tagged agents. In certain embodiments, a plurality ofanti-peptide agents may be present at a ratio in a range of 5:1-10:1, ona mole:mole basis, relative to the plurality of detectable agents, andthe plurality of anti-peptide agents may be present at a ratio of in arange of 200:1-500:1, on a mole:mole basis, relative to a plurality ofpeptide-tagged agents. In certain embodiments, a plurality ofanti-peptide agents may be present at a ratio in a range of 6:1 -1 0:1 ,on a mole:mole basis, relative to the plurality of detectable agents,and the plurality of anti-peptide agents may be present at a ratio of ina range of 250:1-400:1, on a mole:mole basis, relative to a plurality ofpeptide-tagged agents.

In certain embodiments, the analyte complex may comprise, for example,the analyte bound to one peptide-tagged agent. In certain embodiments,the analyte complex may comprise, for example, the analyte bound to aplurality of peptide-tagged agents, for example the analyte bound to 2,3, less than 5, less than 10, or bound to in the range of 10-20peptide-tagged agents. In certain embodiments, the analyte complex mayfurther comprise, for example, the analyte bound to one detectableagent. In other embodiments, the analyte complex may comprise, forexample, the analyte bound to a plurality of detectable agents, forexample the analyte bound to 2, 3, less than 5, less than 10, or boundto in the range of 10-20 detectable agents. For example, the analytecomplex may comprise the analyte bound to 1 peptide-tagged agent and 1detectable agent, or 2 peptide-tagged agents and 1 detectable agent. Incertain embodiments, the analyte complex may comprise, for example, apeptide-tagged agent bound to 2 or more analytes, for example thepeptide-tagged agent bound to 2, 3, less than 5, less than 10, or boundto in the range of 10-20 analytes. In certain embodiments, the analytecomplex may comprise, for example, a detectable agent bound to 2 or moreanalytes, for example the detectable agent bound to 2, 3, less than 5,less than 10, or bound to in the range of 10-20 analytes. For example,the analyte complex may comprise 3 analytes bound to 2 peptide-taggedagents and 1, 2, or 3 detectable agents; or, for example, the analytecomplex may comprise 2 analytes bound to 3 peptide-tagged agents and 6detectable agent.

In certain embodiments, at least one peptide-tagged agent may be capableof binding to a first epitope of the analyte and at least one detectableagents may be capable of binding to a second epitope of the analyte. Incertain embodiments, the type of epitope of the first epitope of theanalyte and the second epitope of the analyte may be the same type ofepitope. In certain embodiments, the type of epitope of the firstepitope of the analyte and the second epitope of the analyte may be twodifferent epitope types. In certain embodiments, the type of epitope ofthe first epitope of the analyte and the second epitope of the analytemay share portions of their sequence identify, for example at least 75%,80%, 85%, 90%, or at least 95% shared sequence identity; or at least75%, 80%, 85%, 90%, or at least 95% shared sequences with theirsequences but not necessarily at the same positions relative to theterminal portions of the sequences. In certain embodiments, the at leastone peptide-tagged agent and the at least one detectable agent may bethe same type of antibody. In certain embodiments, the at least onepeptide-tagged agent and the at least one detectable agent may bedifferent types of antibodies. In certain embodiments, the first epitopeof the analyte and/or the second epitope of the analyte is aphospho-epitope. In certain embodiments, the first epitope of theanalyte and the second epitope of the analyte may overlap in a commonportion of the analyte. In certain embodiments, the first epitope of theanalyte and the second epitope of the analyte may not overlap but may bein close proximity to each other, for example within 50 nm of oneanother, for example within 25 nm, within 10 nm, within 5 nm, within 1nm, or within 5 Angstroms of one anthor. In certain embodiments, thefirst epitope of the analyte and the second epitope of the analyte maybe distal from one another, for example at least 1 nm apart from oneanother, for example at least 2 nm, 5 nm, 10 nm, 25 nm, or at least 50nm apart from one another. In certain embodiments, the first epitope ofthe analyte and the second epitope of the analyte may be in the range of10 Angstroms-10 nm of one another, for example 10 Angstroms-5 nm, 10Angstroms-3 nm, or in the range of 25 Angstroms-3 nm of one another. Incertain embodiments, the first epitope of the analyte and the secondepitope of the analyte do not overlap and may be sufficiently distal toeach such that no steric interactions or substantially no stericinteractions occur between the at least one peptide-tagged agent and theat least one detectable agent. In certain embodiments, the first andsecond epitopes may be bound by the at least one peptide-tagged agentand the at least one detectable agent, respectively.

Certain embodiments may provide an assay platform for detecting ananalyte. In certain embodiments, the assay platform may comprise, forexample, a permeable zone that may be configured, for example, totransport at least a portion of a sample that may comprise the analyte,and optionally a further permeable zone which may be configured, forexample, to receive at least a portion of the sample. In certainembodiments, the permeable zone may comprise, for example, a pluralityof peptide-tagged agents. In certain embodiments, the plurality ofpeptide-tagged agents may be capable of forming an analyte complex thatmay comprise at least one of the plurality of peptide-tagged agents andthe analyte. In certain embodiments, the further permeable zone may beconfigured to receive at least a portion of the portion of the samplefrom the permeable zone. In certain embodiments, the further permeablezone may comprise, for example, a plurality of anti-peptide agentspresent, for example, at a ratio of at least 100:1, one a weight:weightand/or mole:mole basis, relative to the plurality of peptide-taggedagents. In certain embodiments, the plurality of anti-peptide agents maybe configured, for example, to immobilize the at least one of theplurality of peptide-tagged agents. In certain embodiments, theplurality of anti-peptide agents may be configured, for example, toimmobilize the analyte complex (for example, the plurality ofanti-peptide agents may be configured, for example, to immobilize theanalyte complex with a probability of .01-99.95%, for example aprobability of 10-99.95%, 10-25%, 10-50%, 25-75%, 50%-75%, or with aprobability of 75-99.95%). In certain further embodiments, the pluralityof anti-peptide agents may be configured, for example, to immobilize theat least 5% of the plurality of peptide-tagged agents, for example atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or at least99.9% of the plurality of peptide-tagged agents, or all of the pluralityof peptide-tagged agents. In certain embodiments, the plurality ofanti-peptide agents may be configured, for example, to immobilize in therange of 5%-100% of the plurality of peptide-tagged agents, for examplein the range of 10%-95%, 10%-75%, 10%-50%, 25%-95%, 25%-75%, 20%-50%,50%-100%, 50%-75%, 75%-100%, or to immobilize in the range of 75%-99.95%of the plurality of peptide-tagged agents.

In certain embodiments, the permeable zone may be distinguishable fromanother portion of the assay platform, for example the further permeablezone. For example, in certain embodiments, distinguishable features ofthe permeable zone may be inclusive of, but not limited to, any one ormore of the following features: comprising a plurality of peptide-taggedagents; configured to be the portion of the apparatus to which thesample is introduced; configured to be the portion of the apparatus towhich the analyte is introduced; configured to bring the analyte intocontact with the plurality of peptite-tagged agents and/or detectableagents; configured to communicate at least a portion of the sample(and/or a portion of the plurality of peptide-tagged agents, detectableagents, analyte, and/or analyte complex) by passive transport, capillaryaction, wetting, wicking, or a combination of two or more thereof;comprising a material that may be, for example, absorbent, fibrous, orporous; and comprising a release zone configured to introduce at least aportion of the peptide-tagged agents and/or detectable agents into thesample. Other distinguishing features of the permeable zone arecontemplated herein. In certain further embodiments, the anotherportion, for example the further permeable zone, may not comprise one ormore of the foregoing distinguishable features.

In certain embodiments, the further permeable zone may bedistinguishable from another portion of the assay platform, for examplethe permeable zone. For example, in certain embodiments, distinguishablefeatures of the further permeable zone may be inclusive of, but notlimited to, any one or more of the following features: comprising aplurality of anti-peptide agents; configured to receive at least aportion of the sample (and/or a portion of the plurality ofpeptide-tagged agents, detectable agents, analyte, and/or analytecomplex) by passive transport, capillary action, wetting, wicking, or acombination of two or more thereof; comprising a detection zone that maybe configured to be the portion of the apparatus to which the analytecomplex may be immobilized; configured to be the portion of theapparatus to which the analyte may be detected; comprising a materialthat may be, for example, absorbent, fibrous, or porous; and comprisinga membrane, inclusive of, for example, a nitrocellulose membrane. Otherdistinguishing features of the further permeable zone are contemplatedherein.

In certain embodiments, the permeable zone and the further permeablezone may be non-overlapping. In other embodiments, at least a portion ofthe permeable zone and at least a portion of the further permeable zonemay overlap. In certain embodiments, the permeable zone may be containedwithin the further permeable zone. In certain embodiments, the furtherpermeable zone may be contained within the permeable zone.

Certain embodiments may provide a lateral flow device. In certainembodiments, the lateral flow device may comprise, for example, a flowpath that may be defined by, for example, at least a portion of apermeable sub-assembly of the lateral flow device. In certainembodiments, the lateral flow device may further comprise, for example,a release zone that may comprise, for example, a plurality ofpeptide-tagged agents. In certain embodiments, the lateral flow devicemay further comprise, for example, a detection zone that may comprise,for example, a plurality of anti-peptide agents that may be present, forexample, in the flow path at a ratio of at least 100:1, on a weightand/or mole:mole basis, relative to the plurality of peptide-taggedagents. In certain embodiments, the release zone may be configured, forexample, to release at least a portion of said plurality ofpeptide-tagged agents into the flow path. In certain embodiments, atleast one of said plurality of anti-peptide agents may be a bindingpartner with at least one of said plurality of peptide-tagged agents.

In certain embodiments, the permeable sub-assembly may comprise one ormore permeable members, for example, 1, 2, 3, 4, or 5 permeable members.Examples of suitable permeable members are inclusive of, for example,for example, a permeable strip, a fibrous pad, and a porous membrane. Incertain embodiments, one or more of the permeable members may be capableof being wetted. In certain embodiments, one or more of the permeablemembers may comprise matrix of solid material, such as fibers orparticles, interspersed with open spaces. In certain embodiments, theopen spaces may comprise, for example, pores, channels, microchannels,spaces between fibers, cusps formed by adjacent and/or packed particles(for example, particles such as microspheres), and the like. Other typesof open spaces are contemplated herein. In certain embodiments, asample, for example a liquid, suspension, or solution, may be introducedto a portion of the permeable sub-assembly and transported through theopen spaces of at least a further portion of the sub-assembly by passivetransport, capillary action, wetting, wicking, or a combination of twoor more thereof.

In certain embodiments, the flow path may be characterized by the flux,or migration, of a sample being transported through at least a portionof the permeable sub-assembly. In certain embodiments, the flow path maycharacterized by the general, average, or net direction of flowresulting from transport of the sample and/or portions thereof throughthe open spaces in at least a portion of the sub-assembly. In certainembodiments, the flow path may not be uniform in a cross-section of thepermeable sub-assembly. For example, in certain embodiments, a samplemay flow or migrate more quickly through larger open spaces and/or neara boundary of the permeable sub-assembly, for example near or at asurface, for example a top surface, or an edge of the permeablesub-assembly.

In certain embodiments, at least a portion of the flow path may beapproximately parallel, linear, convergent, or divergent. In certainembodiments, at least a portion of the flow path may be a radial flowpath.

In certain embodiments, the release zone may comprise a portion of thepermeable sub-assembly. In certain further embodiments, the plurality ofpeptide-tagged agents may be dried on, bound, releasably bound, orunbound to at least a portion of the permeable sub-assembly. Inembodiments in which at least a portion of the plurality of anti-peptideagents may be unbound to the sub-assembly, the plurality ofpeptide-tagged agents may be introduced to a portion of the sub-assemblywith the sample, or introduced separately from the sample and becomedissolved or suspended in the sample in the open spaces of the permeablesub-assembly. In certain embodiments in which the plurality ofpeptide-tagged agents is bound or releasably bound in the release zone,the release zone may be configured to further release at least a portionof said plurality of peptide-tagged agents into the flow path. Incertain embodiments, at least a portion of the plurality ofpeptide-tagged agents may migrate, dissolve, disconnect, and/or diffusefrom at least a portion of the permeable sub-assembly, for example therelease zone. In certain embodiments, at least a portion of theplurality of peptide-tagged agents may migrate, dissolve, disconnect,and/or diffuse from at least a portion of the permeable sub-assemblyafter contact with the sample and/or a solvent.

In certain embodiments, said plurality of anti-peptide agents may beconfigured, for example, to immobilize at least a fraction of the atleast a portion of said plurality of peptide-tagged agents configured tobe released, for example released in the detection zone of thesub-assembly. In certain further embodiments, at least one of the atleast a portion of said plurality of peptide-tagged agents configured tobe released may be capable of binding with an analyte prior to beingimmobilized by said plurality of anti-peptide agents.

In certain embodiments, the detection zone may comprise a portion of thepermeable sub-assembly. In certain further embodiments, the plurality ofanti-peptide agents may be bound to at least a portion of the permeablesub-assembly. In certain embodiments, the plurality of anti-peptideagents may be passively bound. In certain embodiments, the plurality ofanti-peptide agents may be bound by non-covalent interactions. Examplesof suitable interactions include, for example, electrostaticinteractions, hydrophilic interactions, hydrophobic interactions, ionicelectrostatic interactions, van de Waals interactions, or combinationsof two or more such interactions. In some embodiments, the anti-peptideagent may be actively bound, for example covalently bound, to the solidsubstrate.

In certain embodiments, at least a portion of the permeable sub-assemblymay comprise a plurality of linkers which may facilitate covalentbonding of the plurality of anti-peptide agents. Suitable linkers mayinclude, for example, glutathione, maleic anhydride, a metal chelate, ormeleimide. In certain further embodiments, the plurality of anti-peptideagents may be exposed to at least a portion of the open spaces in atleast a portion of the permeable sub-assembly, for example the openspaces present in the detection zone.

In certain further embodiments, at least a portion of the plurality ofdetectable agents may be dried on, bound, releasably bound, or unboundto at least a portion the permeable sub-assembly. In certain embodimentsin which at least a portion of the plurality of detectable agents may beunbound to the sub-assembly, the detectable agent may be introduced to aportion of the sub-assembly with the sample, or introduced separatelyfrom the sample and become dissolved or suspended in the sample on thepermeable sub-assembly. In certain embodiments in which the plurality ofdetectable agents is bound or releasably bound in the release zone, therelease zone may be configured to further release at least a portion ofsaid plurality of detectable agents into the flow path. In certainfurther embodiments, at least a portion of the plurality of detectableagents may be capable of migrating, dissolving, disconnecting, ordiffusing from at least a portion of the permeable sub-assembly, forexample the release zone. In certain embodiments, at least a portion ofthe plurality of detectable agents may migrate, dissolve, disconnect,and/or diffuse from at least a portion of the permeable sub-assemblyafter contact with the sample and/or a solvent.

Certain embodiments may provide an assembly. In certain embodiments, theassembly may comprise, for example, a permeable release member that mayhave a plurality of peptide-tagged agents deposited on at least aportion thereof and, optionally, a plurality of detectable agentsdeposited on at least a portion thereof. In certain embodiments, theassembly may further comprise, for example, a permeable detection memberthat may be in fluid communication with the permeable release member. Incertain embodiments, the permeable detection member may have a pluralityof anti-peptide agents bound to at least a portion thereof.

In certain embodiments, the permeable release member may absorb at leasta portion of the plurality of peptide-tagged agents and/or at least aportion of the plurality of detectable agents. In certain embodiments,the permeable release member may allow release of at least a portion ofthe plurality of peptide-tagged agents and/or at least a portion of theplurality of detectable agents. In certain embodiments, the permeablerelease member may provide structural support for at least a portion ofthe assembly. In certain embodiments, at least a portion of theplurality of peptide-tagged agents and/or at least a portion of theplurality of detectable agents may be striped onto the permeable releasemember. In certain embodiments, at least a portion of the plurality ofpeptide-tagged agents and/or at least a portion of the plurality ofdetectable agents may be dried onto the permeable release member. Asexplained herein, the striped and/or dried peptide-tagged agents and/ordetectable agents may migrate, dissolve, disconnect, and/or diffuse fromat least a portion of the permeable release member into a sample or asolvent. In certain embodiments, at least a portion of the plurality ofpeptide-tagged agents and/or plurality of detectable agents may be ableto passively mix with a liquid sample present on the permeable releasemember. In certain embodiments, any one or more of the types of analytecomplexes contemplated herein may be able to be formed on the permeablerelease pad.

In certain embodiments, the permeable release member may comprise abibulous, hydrophilic material, such as, for example, an absorbentmaterial. Suitable permeable release member materials may comprise, forexample, cotton linter; cellulosic materials, or materials made ofcellulose together with a polymeric fibrous material, such as polyamideor rayon fibers; and glass fiber material. For example, suitablematerials may include cotton linter paper, such as S&S 903 and S&S GB002(available from Schleicher and Schuell, Inc., Keene, N.H.), and BFC 180(available from Whatman, Fairfield, N.J.); cellulosic materials, such asGrade 939 made of cellulose with polyamide, Grade 989 made of celluloseblend fiber, and Grade 1278 and Grade 1281 made of cellulose and rayonwith polyamide (available from Ahlstrom Corporation, Mt. Holly Springs,Pa.); and glass fiber, such as Lydall borosilicate (available fromLydall, Inc., Rochester, N.H.).

In certain embodiments, the permeable release member may be blocked toprevent non-specific binding. In certain embodiments, the permeablerelease member may be coated with an aqueous solution containing bovineserum albumin (BSA) and a nonionic surfactant, such as polyethyleneglycol, for example the polyethylene glycol sold under the trade nameTriton X-100 (available from Rohm & Haas Co., Philadelphia, Pa.), forexample, a combination of about 3% BSA and about 0.1% Triton X-100.

In certain embodiments, the permeable detection member may be formed ofa substance which may permit binding of the plurality of anti-peptideagents thereto. In certain embodiments, the permeable detection membermay comprise a polymeric material, for example a microporous film ormembrane which may permit at least a portion of the anti-peptide agentsto be passively bound or absorbed thereon. In such embodiments, it maynot be necessary to bind the plurality of anti-peptide agents to thepermeable detection member by chemical or physical fixation. In otherembodiments, the plurality of anti-peptide agents may be bound to thepermeable detection member by chemical or physical fixation.

In certain embodiments, suitable permeable detection member materialsmay comprise, for example, a microporous polymeric film ofnitrocellulose, nylon, charge-modified nylon, polyvinylidine fluoride,polyethersulfone, or similar materials, or combinations of suchmaterials. In certain embodiments, suitable permeable detection membermaterials may have a pore size in a range of between 0.1 μm and 20 μm,for example in a range of between 1 μm and 3 μm, 3 μm and 10 μm, 10 μmand 20 μm, or in a range of between 5 μm and 20 μm.

In certain embodiments, the permeable detection member may comprise anitrocellulose membrane, inclusive of, for example, a nitrocellulosemembrane having added detergents and/or surfactants. In certainembodiments, the permeable detection member may comprise anitrocellulose membrane, inclusive of, for example, a membranecomprising nitrocellulose alone or a mixed ester of nitrocellulose, suchas in combination with an ester of nitric acid and/or other acids. Incertain further embodiments, a nitrocellulose membrane permeabledetection member may be coated or laminated onto a translucent ortransparent polymeric film to provide physical support for the membrane.In certain embodiments, the permeable detection member may comprise anitrocellulose polymer which has been cast onto a polyester film, suchas MYLAR®. In certain embodiments, the permeable detection member maycomprise a nitrocellulose membrane laminated onto a polyester film, orlaminated onto another backing materials besides polyester. Suitablebacking materials may include, for example, pre-laminated or pre-castplanar sheets.

In certain embodiments, the permeable release member and the permeabledetection member may be joined by overlapping a downstream edge of thepermeable release member over an upstream edge of the permeabledetection member, then adhering the resulting biphasic material to aclear polymer film or sheet, thereby holding the two members in place.In other embodiments, the permeable release member and the permeabledetection member may be joined at a non-overlapping butt-joint, andadhered to a clear polymer film or sheet, thereby holding the twomembers in place. In certain embodiments, the joined members may providea continuous flow path for transport of the sample, peptide-taggedagent, detectable agent, analyte, analyte complex, or any combination ofthe foregoing. Transport may comprise, for example, passive transport,capillary action, wetting, wicking, or a combination of two or morethereof. In certain embodiments, the joined members are in fluidcommunication.

In certain embodiments, at least one of the permeable release member andthe permeable detection member has at least one flow channel spanning atleast a portion of the respective member with a smallest cross-sectionalwidth in a range of 3-20 times a diameter of a sphere having a molecularvolume of the analyte complex. In certain embodiments, the at least oneflow channel has a smallest cross-section width of less than 10, from 10to 20, or greater than 20 times a diameter of a sphere having amolecular volume of the analyte complex.

A schematic view of a representative lateral flow detection apparatus100 is illustrated in FIG. 1. According to this embodiment, absorbentsample pad 106, permeable release member 108, permeable detection member110, and absorbent wick pad 112 are affixed onto backing card 102 intofluid communication. A liquid sample containing an analyte may beintroduced to absorbent sample pad 106 and fluidly communicated bycapillary action to permeable release member 108 where the analyte mayencounter at least one peptide-tagged agent and, optionally, adetectable agent in the liquid sample, at which time it may bind to forman analyte complex. The analyte complex may then be fluidly communicatedthrough the remainder of permeable release member 108 and be introducedto permeable detection member 110. The analyte complex will continue toflow in permeable detection member 110 unless and until it isimmobilized thereon. A portion of the liquid sample, includingpotentially non-immobilized analyte complex, will flow through permeabledetection member 110 and be absorbed into wick pad 112.

A schematic view of solvent-driven lateral flow detection apparatus 200is illustrated in FIG. 2. According to this embodiment, absorbentsolvent pad 204, permeable sample pad 206, permeable release member 208,permeable detection member 210, and absorbent wick pad 212 are affixedonto backing card 202 into fluid communication. A liquid solvent may beintroduced to absorbent solvent pad 204 and fluidly communicated bycapillary action to permeable sample pad 206 where the solvent maydissolve or suspend an analyte present in the sample. Theanalyte-containing solvent may then flow to permeable release member 208where the analyte may encounter at least one peptide-tagged agent and,optionally, a detectable agent in the solvent, at which time the analytemay bind to form an analyte complex. The analyte complex may then befluidly communicated through the remainder of permeable release member208 and be introduced to permeable detection member 210. The analytecomplex will continue to flow in permeable detection member 210 unlessand until it is immobilized thereon. A portion of the solvent, includingpotentially non-immobilized analyte complex, will flow through permeabledetection member 210 and be absorbed into wick pad 212.

A schematic view of multi-purpose lateral flow test kit 300 configurableto detect any one of three types of analytes is illustrated in FIG. 3.According to this embodiment, absorbent sample pad 306, universalreceiver for a permeable release member 308, permeable detection member310, and absorbent wick pad 312 are affixed onto backing card 302,bringing permeable detection member 310, and absorbent wick pad 312 intofluid communication. First permeable release member 308A, secondpermeable release member 308B, or third permeable release member 308C,each optionally containing a different type of peptide-tagged agentand/or detectable agent, may be inserted into universal receiver 308 tobring the inserted member into fluid communication with absorbent samplepad 306 and permeable detection member 310.

A schematic view of representative radial flow detection apparatus 400for the detection of eight (8) different analytes is illustrated in FIG.4. According to this embodiment, absorbent sample pad 406 is centrallypositioned and in fluid communication with permeable release member 408,permeable detection member 410, and absorbent wick pad 412. A liquidsample optionally containing a plurality of different types of analytesmay be introduced to absorbent sample pad 406 and flow radially(divergently) by capillary action to permeable release member 408 whereeach of the analytes may encounter at least one peptide-tagged agent towhich it can bind and a detectable agent to which it can bind in theliquid sample, at which time it may bind to form an analyte complex. Theanalyte complex may then be fluidly communicated through the remainderof permeable release member 408 and be introduced to permeable detectionmember 410. The analyte complex will continue to flow in permeabledetection member 410 unless and until it is immobilized thereon. Aportion of the liquid sample, including potentially non-immobilizedanalyte complex, will flow through permeable detection member 410 and beabsorbed into wick pad 412. In order to detectably distinguish betweeneach of the up to eight types of analytes that may be present in thesample, apparatus 400 may be divided into eight sections, as delineatedby dashed lines 414A-H. Within each section, a different plurality ofpeptide-tagged agents and a different plurality of detectable agents maybe striped onto the portion of release zone 408 present in each section,forming stripes 408A-H. Within each section, the different plurality ofpeptide-tagged agents and the different plurality of detectable agentsmay be striped together, in sequence, or side by side relative to thedirection of flow of the sample. Within each section, a plurality ofanti-peptide agents may be striped onto the portion of detection zone410 present in each section, forming stripes 410A-H. In certainexemplary embodiments, the type of anti-peptide agent present in eachsection may be the same. In other exemplary embodiments, different typesof anti-peptide agents may be used in two or more of the sections. Incertain embodiments, one or more of the sections may be in fluidcommunication. In certain embodiments, one or more of the sections maynot be in fluid communication, for example one or more of the sectiondelimiters indicated by dashed lines 414A-H may also denote the presenceof a fluid barrier (e.g., a plastic wall) which prevents fluidcommunication between two or more sections. As is apparent from FIG. 4,in certain embodiments the radial flow detection apparatus may bereadily configured to have fewer or more than eight (8) sections, andmay thereby be configured to detect fewer or more than eight (8)different types of analytes.

A schematic view of a representative multi-analyte lateral flowdetection apparatus 500 for the detection of six (6) different analytesis illustrated in FIG. 5. According to this embodiment, absorbent samplepad 506, permeable release member 508, permeable detection member 510,and absorbent wick pad 512 are affixed onto backing card 502 into fluidcommunication. A liquid sample optionally containing a plurality ofdifferent types of analyte may be introduced to absorbent sample pad 506and fluidly communicated by capillary action to permeable release member508 where each of the analytes may encounter at least one peptide-taggedagent to which it may bind and a detectable agent to which it may bindin the liquid sample, at which time it may bind to form an analytecomplex. Each of the analyte complexes formed thereby may then befluidly communicated through the remainder of permeable release member508 and be introduced to permeable detection member 510. Each analytecomplex will continue to flow in permeable detection member 510 unlessand until it is immobilized thereon. A portion of the liquid sample,including potentially non-immobilized analyte complex, will flow throughpermeable detection member 510 and be absorbed into wick pad 512.Permeable release member 508 is striped with a plurality of six (6)types of peptide-tagged agents and with six (6) types of detectableagents in order to provide the types of agents required to bind witheach of the six (6) types of analytes. The plurality of six (6) types ofpeptide-tagged agents and the plurality of six (6) types of detectableagents may be striped together in zone 508A. Alternatively, theplurality of six (6) types of peptide-tagged agents may be striped inzone 508A and the plurality of six (6) types of detectable agents may bestriped together in zone 508B. Permeable detection member 510 may bestriped with a plurality of up to six (6) or more types of anti-peptideagents, each capable of immobilization at least one of the types ofpeptide-tagged agents. The plurality of six (6) or more types ofanti-peptide agents may be striped together in zone 510A. Alternatively,one or more additional zones 510B-F may be used to separately strip oneor more of the plurality of six (6) or more types of anti-peptideagents. Additionally, permeable detection member 510 may comprisecontrol zone 510G configured to provide an indication that the apparatushas functioned, for example by detectably capturing any type of particlepresent in the sample. As is apparent from FIG. 5, in certainembodiments the multi-analyte lateral flow detection apparatus may bereadily configured to detect fewer or more than six (6) analytes byadjusting the number and types of peptide-tagged agents, detectableagents, and anti-peptide agents and the striping scheme.

EXAMPLES Lateral Flow Test Strip Preparation

Release Pads: Millipore GFDX glass fiber pads were blocked with blockingbuffer (10 mM borate, 3% BSA, 1% PVP-40, 0.25% Tx-100), dried, andstored desiccated. The dried glass fiber pads were then sprayed withdetectable agent and, optionally, a peptide-tagged agent, in accordancewith Table 1 and then dried at approximately 40° C. and storeddesiccated.

Detection Membranes: Sartorius Unisart CN140 nitrocellulose membraneswere striped with an immobilization agent in accordance with Table 1using a Biodot frontline dispenser at a dispense rate of about 1 pL/cm,dried, and stored dessicated. The nitrocellulose membranes were thenblocked with blocking buffer (10mM phosphate, 0.1% sucrose, 0.1% BSA,0.2% PVP-40, pH 7.2), dried at approximately 40° C., and storeddesiccated.

TABLE 1 Conjugate Pad and Detection Membrane Preparations DetectionConjugate Pad Membrane Prepa- Peptide-Tagged Detectable Immobilizationration Agent Agent Agent A 6.5 ng anti-hCG-β 250 ng anti- 2000 ng mousetagged with a plurality hCG-α monoclonal IgG1 of peptide tags conjugatedto immobilization agent (sequence: colloidal gold (capable of CDYKDDDDKimmobilizing the (SEQ ID NO: 8)) tagged anti-hCG- β) B (no Capture Agent500 ng anti- 500 ng anti-hCG-β was placed hCG-α (capable of directlyConjugate Pad) conjugated to immobilizing hCG) colloidal gold

Lamination and Assembly: Each lateral flow test strip comprised: (1) aconjugate pad having a 2 mm overlap with the top of one end of adetection membrane, (2) a wick pad having a 2 mm overlap with the top ofthe opposite end of the detection membrane, and (3) an adhesive backingcard onto which the conjugate pad—detection membrane—wick padconfiguration was affixed. 160 test strips were used (80 each of basedon Preparation A and Preparation B, respectively) in the followingExample.

Example 1: A series of hCG detection experiments were conducted at theconcentrations shown in Table 2. In each experiment, 40 pL of hCGsolution was deposited on the conjugate pad and allowed to run for 8minutes. Afterwards, a wash solution was deposited on the conjugate padand allowed to run for 7 minutes. Results were obtained by detection ofdetectable agent present in the test zone with an ESE TS012 Gold Readerand reported in Table 2.

TABLE 2 Detection Results for Example 1 Lateral Flow Test Strip BasedLateral Flow Test Strip Based On On Preparation A Preparation BExperiment Concentration Average Standard Average Standard No. hCG,mIU/mL Signal* Deviation Signal/Noise Signal* Deviation Signal/Noise 1 08.3 4.96 1.00 16.4 9.99 1.00 2 0.25 13.9 5.82 1.67 14.2 7.51 0.87 3 0.516.5 6.41 1.99 20.0 5.16 1.22 4 1 16.2 6.71 1.95 13.0 6.28 0.80 5 2 33.312.60 4.01 19.3 6.75 1.18 6 5 71.9 17.45 8.66 49.4 7.59 3.02 7 10 112.415.01 13.54 90.0 7.22 5.51 8 15 164.3 20.42 19.80 121.5 14.89 7.43 9 25197.7 32.45 23.82 177.3 24.34 10.84 10 50 349.8 35.40 42.14 279.5 11.8717.10 11 100 451.5 65.08 54.39 389.1 20.75 23.80 *Each experiment wasrepeated 4-8 times resulting in a total of 80 experiments each forLateral Flow Test Strip Preparation A and Preparation B, respectively.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

While certain embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is intendedthat the following claims define the scope of the invention and thatmethods and structures within the scope of these claims and theirequivalents be covered thereby.

1. A method to detect a pre-determined analyte, comprising: i) exposinga sample comprising the pre-determined analyte to a plurality ofpeptide-tagged agents to form an analyte complex, said analyte complexcomprising the pre-determined analyte and at least one of the pluralityof peptide-tagged agents; and ii) transporting the analyte complex to adetection zone of a lateral flow device, said detection zone comprisinga plurality of anti-peptide agents present at a ratio of at least 100:1,on a weight:weight basis, relative to the plurality of peptide-taggedagents, said plurality of anti-peptide agents capable of immobilizing atleast one of the at least one of the plurality of peptide-tagged agents;and iii) detecting the pre-determined analyte.
 2. The method of claim 1,wherein the transporting comprises capillary flow action along a flowpath.
 3. The method of claim 2, wherein the flow path is a divergentflow path.
 4. The method of claim 1, wherein the plurality ofpeptide-tagged agents is releasably bound to a release zone of thelateral flow device.
 5. The method of claim 1, further comprising:introducing the analyte complex to the lateral flow device.
 6. Themethod of claim 1, wherein the plurality of anti-peptide agents ispresent at a ratio of at least 300:1, on a weight:weight basis, relativeto the plurality of peptide-tagged agents.
 7. The method of claim 1,further comprising: immobilizing the analyte complex in the detectionzone of the lateral flow device.
 8. The method of claim 7, whereindetecting the pre-determined analyte comprises detecting the immobilizedanalyte complex.
 9. The method of claim 1, further comprising: exposingsaid sample to a plurality of detectable agents, said analyte complexfurther comprising at least one of said plurality of detectable agents.10. The method of claim 9, further comprising: immobilizing the analytecomplex in the detection zone; wherein detecting the pre-determinedanalyte comprises detecting the at least one of said plurality ofdetectable agents.
 11. The method of claim 9, wherein the plurality ofdetectable agents and the plurality of peptide-tagged agents are mixedand striped onto a release zone of the lateral flow device.
 12. Themethod of claim 9, wherein the plurality of detectable agents and theplurality of peptide-tagged agents are separately striped onto therelease zone.
 13. The method of claim 9, wherein said plurality ofanti-peptide agents are present at a ratio of at least 8:1, on aweight:weight basis, relative to the plurality of detectable agents. 14.The method of claim 9, wherein the plurality of detectable agents arepresent at a ratio of at least 2:75, on a weight:weight basis, relativeto the plurality of peptide-tagged agents.
 15. The method of claim 1,wherein at least one of the plurality of peptide-tagged agents comprisesa plurality of covalently-conjugated peptide tags.
 16. The method ofclaim 15, wherein at least one of the plurality of anti-peptide agentscomprises a specific binding partner to at least one of the plurality ofcovalently conjugated peptide tags.
 17. The method of claim 1, whereinthe sample is formed by contacting a solvent with a material present onthe lateral flow device.
 18. The method of claim 17, wherein the solventis contacted with the material present on the lateral flow device.
 19. Amethod to detect a pre-determined analyte, comprising: i) receiving asample comprising the pre-determined analyte to a permeable release zoneof a lateral flow device, said permeable release zone comprising aplurality of peptide-tagged agents and a plurality of detectable agentsstriped thereon; ii) forming an analyte complex comprising thepre-determined analyte bound to at least one of the plurality ofpeptide-tagged agents and separately bound to at least one of theplurality of detectable agents, wherein the at least one of theplurality of peptide-tagged agents: a) is a pre-determined specificbinding partner with the pre-determined analyte; and b) comprises aplurality of covalently conjugated peptide tags; iii) transporting theanalyte complex by capillary flow action to a detection zone of thelateral flow device, said detection zone comprising a plurality ofanti-peptide agents, wherein said plurality of anti-peptide agents arepresent at a ratio of: a) at least 300:1, on a weight:weight basis,relative to the plurality of peptide-tagged agents; and b) at least 8:1,on a weight:weight basis, relative to the plurality of detectableagents; iv) immobilizing the analyte complex to the detection zone byselectively binding at least one of the plurality of anti-peptide agentswith at least one of the plurality of covalently conjugated peptidetags; and v) detecting the immobilized analyte complex within 8 minutesof introducing the sample.
 20. (canceled)
 21. The method of claim 3,wherein: i) the plurality of peptide-tagged agents is releasably boundto a central release zone of the lateral flow device; and ii) thedivergent flow path is a radial flow path.